ES2338111T3 - PIG ANIMALS THAT LACK OF ANY EXPRESSION OF ALPHA 1.3 FUNCTIONAL GALACTOSILTRANSPHERASE. - Google Patents

Abstract

The present invention is a porcine animal, tissue, organ, cells and cell lines, which lack any expression of functional alpha 1,3 galactosyltransferase (alpha 1,3GT). These animals, tissues, organs and cells can be used in xenotransplantation and for other medical purposes.

Description

Swine animals that lack any expression of functional alpha 1,3 galactosyltransferase.

This request claims priority to the U.S. Provisional Patent Application No. 60 / 404,775 filed on August 21, 2002.

Field of the Invention

The present invention are animals, tissues and porcine organs as well as cells and cell lines obtained from said animals, tissues and organs, which lack any Functional alpha 1,3 galactosyltransferase (alpha1,3GT) expression. Such animals, tissues, organs and cells can be used in research and medical therapy, including in Xenotransplant

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Background of the invention

Patients with organic failure in the final phase They require organ transplantation for survival. He main limiting factor in clinical transplantation is the shortage of suitable human donors. Over the past ten years patient waiting list has increased dramatically for organs, from about 30,000 in 1991 to about 80,000 in 2001 (source: The New York Organ Donor Network (New York Organ Donor Network); Registry Review Study Deaths of the Association of Organizations for the Recovery of Organs (Association of Organ Procurement Organizations' Death Record Review Study) from 1997 to 1999, provided by 30 organizations for organ recovery). Despite this growing need over the past ten years, the amount of organ donations has remained fixed (approximately 20,000 per year).

According to the International Network of Organ Distribution (United Network for Organ Sharing) (UNOS) dated July 17, 2003, there were 82,249 patients waiting organ transplants in the United States. The need to Specific organs is as follows:

one

Across the United States, an average of 17 men, women and children of all races and ethnic backgrounds they die every day due to the absence of donated organs, therefore, each year, more than 6,200 Americans die waiting for a transplant of organ. The need for a more reliable and unlimited source of organs has led to the investigation of the potential for transplantation of organs of other animals, called xenotransplantation.

Pigs are considered the most likely source of organs for xenograft. The supply of pigs is abundant, breeding programs are well established, and their size and physiology are compatible with humans. The xenotransplant, however, presents its own series of problems. The most significant is immune rejection. The first immune obstacle is "hyperacute rejection" (HAR). HAR can be defined by the ubiquitous presence of high levels of preformed natural antibodies that bind to foreign tissue. It is believed that the binding of these natural antibodies to target epitopes in the endothelium of the donor organ is the initial event in the HAR. This union, in minutes from the perfusion of the donor organ with the blood of the recipient, is followed by activation of the complement, deposition of platelets and fibrin, and finally by interstitial edema and hemorrhage in the donor organ, all of which causes organ failure. the recipient (Strahan et al . (1996) Frontiers in Bioscience 1,
e34-41).

Except for old world monkeys, apes and humans, most mammals carry glycoproteins on their cell surfaces that contain galactose alpha 1,3-galactose (Galili et al ., J. Biol. Chem. 263: 17755 -17762, 1988). Humans, apes and old-world monkeys have a naturally occurring anti-alpha gal antibody that is produced in large quantities (Cooper et al ., Lancet 342: 682-683, 1993). It specifically binds to glycoproteins and glycolipids that harbor galactose alpha-1,3 galactose.

In contrast, glycoproteins containing galactose alpha 1,3-galactose are found in large amounts in cells of other mammals, such as pigs. This differential distribution of the "alpha-1,3 GT epitope" and anti-Gal antibodies (ie, antibodies that bind glycoproteins and glycolipids that harbor galactose alpha-1,3 galactose) in mammals is the result of an evolutionary process that selected species with inactivated alpha-1,3-galactosyltransferase (i.e. mutated) in ancient world ancestral primates and humans. Therefore, humans are "natural knockouts" of alpha1.3GT. A direct consequence of this event is the rejection of xenografts, such as the rejection of pig organs transplanted in humans initially by
HAR.

A variety of strategies have been carried out to eliminate or modulate the humoral anti-Gal response caused by porcine xenotransplantation, including enzymatic removal of the epitope with alpha-galactosidases (Stone et al ., Transplantation 63: 640-645, 1997), elimination of the specific anti-gal antibody (Ye et al ., Transplantation 58: 330-337,1994), coating the epitope with other carbohydrate residues, which failed to eliminate the expression of alpha-1,3-GT (Tanemura et al. ., J. Biol. Chem. 27321: 16421-16425, 1998 and Koike et al ., Xenotransplantation 4: 147-153, 1997) and the introduction of complement inhibitor proteins (Dalmasso et al ., Clin. Exp. Immunol. 86: 31-35, 1991, Dalmasso et al . Transplantation 52: 530-533 (1991)). C. Costa et al . (FASEB J 13, 1762 (1999)) reported that competitive inhibition of alpha-1,3-GT in transgenic pigs for H-transferase caused only partial reduction in epitope amounts. Also, S. Miyagawa et al . (J Biol. Chem 276, 39310 (2001)) reported that attempts to block the expression of gal epitopes in transgenic pigs for N-acetylglucosaminyltransferase III also caused only partial reduction of gal epitope amounts and failed to significantly prolong the transplant survival in primate recipients.

Knockouts of a single allele of the alpha-1,3-GT locus in pig cells and live animals have been reported. Denning et al . (Nature Biotechnology 19: 559-562, 2001) reported the directed gene deletion of an allele of the alpha-1,3-GT gene in sheep. Harrison et al . (Transgenics Research 11: 143-150, 2002) reported the production of heterozygous knockout somatic porcine fetal fibroblastic cells for alpha-1,3-GT. In 2002, Lai et al . (Science 295: 1089-1092, 2002) and Dai et al . (Nature Biotechnology 20: 251-255, 2002) reported the production of pigs, in which an allele of the alpha-1,3-GT gene became satisfactorily inactive. Ramsoondar et al . (Biol of Reproduc 69, 437-445 (2003)) reported the generation of heterozygous knockout pigs for alpha-1,3-GT that also express human alpha-1,2-fucosyltransferase (HT), which expressed both epitopes both HT as alpha-1,3-GT.

PCT Publication No. WO 94/21799 and the patent of United States No. 5,821,117 of the Austin Research Institute; the PCT Publication No. WO 95/20661 by Bresatec; and PCT publication Nº WO 95/28412, U.S. Patent No. 6,153,428, the patent of the United States Nº 6,413,769 and the publication of the United States Nº 2003/0014770 from BioTransplant, Inc. and The General Hospital Corporation provide an analysis of cell production negative alpha-1,3-GT swine in based on the knowledge of the cDNA of the gene of the alpha-1,3-GT (and without the knowledge of the organization or genomic sequence). However, there was no evidence that such cells actually occurred before the date of submission of these applications and the examples are All prophetic.

The first public description of the successful production of a heterozygous negative alpha-1,3-GT porcine cell occurred in July 1999 at the Lake Tahoe Transgenic Animal Conference (David Ayares, et al ., PPL Therapeutics, Inc.). Prior to the present invention, no one had published or publicly described the production of a homozygous negative alpha 1.3GT swine cell. In addition, since swine embryonic stem cells have not been available to date, there was and there is still no way to use a homozygous embryonic stem cell for alpha-1,3-GT to try to prepare a knockout pig for alpha-1,3- Live homozygous GT.

On February 27, 2003, Sharma et al . (Transplantation 75: 430-436 (2003)) published a report demonstrating a satisfactory production of homozygous fetal fibroblastic pig cells for the elimination of the alpha-1,3-GT gene.

PCT Publication No. WO 00/51424 of PPL Therapeutics describes the genetic modification of somatic cells for nuclear transfer. This patent application describes the genetic alteration of the gene of the alpha-1,3-GT in somatic cells pigs, and the subsequent use of the nucleus of these cells that lack of at least one copy of the gene from the alpha-1,3-GT for transfer nuclear.

U.S. Patent No. 6,331,658 of Cooper and Koren claims but does not confirm any real production of mammals designed by genetic engineering that express a Sialyltransferase or fucosyltransferase protein. The patent states that mammals designed by genetic engineering would show a reduction of galactosylated protein epitopes on the surface mammalian cell

PCT Publication No. WO 03/055302 of The Curators of the University of Missouri confirms the production of knockout pigs for heterozygous alpha 1.3GT for use in xenotransplantation. This application is directed in general lines to knockout pigs that include an altered alpha-1,3-GT gene, where the expression of functional alpha-1,3-GT in knockout pigs is decreased compared to the wild type. This request does not provide any guidelines as to how much alpha-1,3-GT should be decreased so that pigs are useful for xenotransplantation. In addition, this application does not provide any evidence that the heterozygous pigs that were being produced showed a decreased expression of functional alpha1.3GT. In addition, although the request refers to homozygous alpha 1.3GT knockout pigs, there is no evidence in the request that any was actually occurring or could occur, much less if the resulting offspring would be viable or phenotypically useful for xenotransplantation.

The total elimination of glycoproteins that contain galactose alpha 1,3-galactose is clearly the best approach to pig animal production for xenotransplantation. Theoretically it is possible for double knockouts, or the alteration of both copies of the 1,3GT alpha gene, could be produced by two methods: 1) mating of two animals knockout of a single allele to produce offspring, in whose case, it would be predicted based on Mendelian genetics that one of every four would be double knockout or 2) genetic modification of the second allele in a cell with a single elimination preexisting. In fact, this has been quite difficult, as illustrated by the fact that although the first request for Knockout swine cell patent was filed in 1993, the first Homozygous alpha 1.3GT knockout pig did not occur until July 2002 (which was based on the work of the present inventor and described in this document).

Transgenic mice (not pigs) have historically been the preferred model to study the effects of genetic modifications on mammalian physiology, for several reasons, of which mouse embryonic stem cells have been available while porcine embryonic cells have not They have been available is not the minimum. Mice are ideal animals for basic research applications because they are relatively easy to manipulate, reproduce quickly, and can be genetically manipulated at the molecular level. Scientists use mouse models to study the molecular pathologies of a variety of genetically based diseases, from colon cancer to mental retardation. Thousands of genetically modified mice have been created to date. A "mouse knockout and mutation database" has been created by BioMedNet to provide an extensive database of phenotypic and genotypic information about knockouts and classical mutations in mice ( http: // research .bmn.com / mkmd ; Brandon et al . Current Biology 5 [7]: 758-765 (1995); Brandon et al . Current Biology 5 [8]: 873-881 (1995)), this database provides information over more than 3,000 unique genes, which have been modified in a targeted way in the mouse genome to date.

Based on this extensive experience with mice, it has been learned that transgenic technology has some significant limitations. Because of developmental defects, many genetically modified mice, especially null mice created by gene knockout technology die as embryos before the researcher has a chance to use the model for experimentation. Even if mice survive, they can develop significantly altered phenotypes, which can make them severely disabled, deformed or weakened (Pray, Leslie, The Scientist 16 [13]: 34 (2002); Smith, The Scientist 14 [15]: 32, ( 2000); Brandon et al . Current Biology 5 [6]: 625-634 (1995); Brandon et al . Current Biology 5 [7]: 758-765 (1995); Brandon et al . Current Biology 5 [8]: 873-881 (1995); http://research.bmn.com/mkmd ) Furthermore, it has been learned that it is not possible to predict whether or not a given gene plays a critical role in the development of the organism and, therefore, if the elimination of the gene will cause a lethal or altered phenotype, until the knockout has been successfully created and viable offspring have been produced.

Mice have been genetically modified to eliminate functional alpha-1,3-GT expression. Double knockout mice have been produced for alpha-1,3-GT. They are embryologically viable and have normal organs (Thall et al . J Biol Chem 270: 21437-40 (1995); Tearle et al . Transplantation 61: 13-19 (1996), see also U.S. Patent No. 5,849,991) . However, two phenotypic abnormalities were evident in these mice. First, all mice develop dense cortical cataracts. Second, the elimination of both alleles of the alpha-1,3-GT gene significantly affected the development of mice. Mating heterozygous mice for the alpha-1,3-GT gene produced genotypic proportions that deviated significantly from the predicted 1: 2: 1 Mendelian ratio (Tearle et al . Transplantation 61: 13-19 (1996)).

Pigs have a level of cell surface glycoproteins containing galactose alpha 1,3-galactose that is 100-1000 times higher than that found in mice. (Sharma et al . Transplantation 75: 430-436 (2003); Galili et al . Transplantation 69: 187-190 (2000)). Therefore, alpha 1,3-GT activity is more critical and more abundant in pigs than in mice.

Despite predictions and prophetic claims, before this invention, no one knew if the alteration of both alleles of the alpha-1,3-GT gene would be lethal or affect pig development or cause an altered phenotype (Ayares et al Graft 4 (1) 80-85 (2001); Sharma et al . Transplantation 75: 430-436 (2003); Porter and Dallman Transplantation 64: 1227-1235 (1997); Galili, U. Biochimie 83: 557-563 (2001)). In fact, many experts in the field expressed serious doubts about whether homozygous alpha-1,3-GT knockout pigs would be entirely viable, much less if they would develop normally. Such concerns were expressed until the double knockout pig of the present invention occurred. Below are examples of statements by those currently working in the field.

"The expressed alpha-gal epitope you can abundantly have some biological roles in the pig development, such as in interaction cell-cell If the assumption is correct, you can that pigs do not develop in the absence of this epitope (Galili, U. Biochimie 83: 557-563 (2001) ".

"The inability to generate knockout pigs for alpha-gal may suggest that alpha-gal epitopes are indispensable in this species (Galili et al . Transplantation 69: 187-190 (2000))."

"Although double knockout mice for alpha-gal can be developed and maintained quite normal, there is a possibility that the deletion of this enzyme have more serious consequences on other animals (Porter and Dallman Transplantation 64: 1227-1235 (1997)) ".

"It is possible that the GT pig (- / -) may not be viable because the GT gene is essential for embryonic development. An answer to this question and the relevance of GT pigs (- / -) to the search for Xenotransplants should wait, if possible, for the production of the appropriate pigs (Sharma et al . Transplantation 75: 430-436 (2003)) ".

"Since the expression of the Gal epitope in pig organs is up to 500 times greater than in mouse organs, there is a possibility that alphaGT activity is more crucial for the pig and could affect the development of these pigs (Ayares et al . Graft 4 (1) 80-85 (2001)) ".

Therefore, until a pig is produced double knockout for the alpha-1,3-GT viable, according to current technical specialists, not it will be possible to determine (i) if the offspring would be viable or (ii) if the offspring would present a phenotype that allows the use of organs for transplantation in humans.

Therefore, an object of the present invention is to provide viable pigs that lack any expression of functional alpha1,3GT.

Another object of the present invention is provide porcine cells, tissues and organs that lack any expression of functional alpha1,3GT, for use in Xenotransplantation or other biomedical applications.

A further object of the present invention is provide a method to select and detect cells swine, lacking epitopes galactose alpha 1,3-galactose on the cell surface.

Summary of the invention

The invention provides a pig in which both alleles of the gene of the alpha-1,3-GT are inactive, where a allele becomes inactive through at least one mutation punctual.

This invention is the production of the first live pigs that lack any functional expression of alpha 1,3 galactosyltransferase. The object of this invention was announced in full page in the journal Science in 2003 (Phelps et al . (Science 299: 411-414 (2003)) and was widely presented in the press as a major advance in xenotransplantation.

It has been shown for the first time that it can produce a viable pig animal that lacks any expression of functional alpha 1,3 galactosyltransferase. The present invention provides complete inactivation of both alleles of the alpha 1,3 galactosyltransferase gene in pigs, exceeding this way this old obstacle and making the xenotransplant a reality. The elimination of this gene, which causes the absence of galactose alpha 1,3-galactose epitopes on the cell surface, represents the first and main stage in the elimination of hyperacute rejection in xenotransplant therapy pig-to-human being. The invention also provides organs, tissues, and cells obtained from said pig animals, which are useful for xenotransplantation.

In embodiments of the present invention, the alleles of the alpha-1,3-GT gene are they become inactive, where an allele becomes inactive through the minus a point mutation, so that the enzyme resulting alpha-1,3-GT can no longer generate galactose alpha 1,3-galactose on the cell surface In one embodiment, the gene of the alpha-1,3-GT can be transcribed in RNA, but cannot be translated into protein. In another embodiment, the alpha-1,3-GT gene can Transcribe in an inactive truncated form. Said truncated RNA it may not translate or it may translate into a protein not functional. In an alternative embodiment, the gene of the alpha-1,3-GT can be inactivated from such that gene transcription does not happen. In one embodiment additional, the alpha-1,3-GT gene can be transcribed and then translated into a protein not functional.

Pigs that lack any expression of functional alpha-1,3-GT are useful to provide a clearer evaluation of the approaches currently in development focused on overcoming the mechanisms of potential delayed and chronic rejection in xenotransplantation porcine.

At least one allele of the gene of the alpha-1,3-GT can be inactivated through an event of directed genetic modification. In another embodiment of the present invention, both alleles of the gene of the alpha-1,3-GT are inactivated through an event of directed genetic modification. The Gen can be modified in a directed way by recombination homologous In other embodiments, the gene can be altered, that is, part of the genetic code may be altered, affecting this mode to transcription and / or translation of that segment of the gene. By For example, an alteration of a gene can happen through techniques replacement, deletion ("knockout") or insertion ("knockin"). Additional genes can be inserted for a desired protein or a regulatory sequence that modulates the transcription of an existing sequence.

Pigs that have two inactive alleles of the alpha-1,3-GT gene are not from natural origin The predicted frequency of incidence of said pig it would be in the range of 10-10 to 10-12, and it has never been identified.

Surprisingly it was discovered that while tried to eliminate the second allele of the gene from the alpha-1,3-GT through a directed genetic modification event, a point mutation, which returned to the second inactive allele. Pigs that carry point mutations in the gene of the alpha-1,3-GT are gene free of antibiotic resistance and therefore have the potential to Create a safer product for human use. Therefore, the pig of the invention can be a homozygous knockout for alpha-1,3-GT that has no genes for resistance to antibiotics or other selection marker genes. In one embodiment, this point mutation can happen through a directed genetic modification event. In other realization, this point mutation can happen naturally. In a further embodiment, mutations in the gene can be induced. of the alpha-1,3-GT using an agent mutagenic

In a specific embodiment, the point mutation may be a TaG mutation at the second base of exon 9 of the alpha-1,3-GT gene (Figure 2). In other embodiments, there may be at least two, at least three, at least four, at least five, at least ten or at least twenty point mutations to return to the inactive alpha-1,3-GT gene. In other embodiments, pigs are provided in which both alleles of the alpha-1,3-GT gene contain point mutations that prevent any expression of functional alpha1.3GT. In a specific embodiment, pigs containing the TaG mutation at the second base of exon 9 are provided in both alleles of the alpha-1,3-GT gene
(Figure 2).

Another embodiment of the present invention provides a pig animal, in which both alleles of the gene of the alpha-1,3-GT are inactivated, by which an allele is inactivated by a point mutation. In a embodiment, a pig animal is provided, in which both alleles of the alpha-1,3-GT gene are inactivated, whereby an allele is inactivated by a directed genetic modification event and the other allele is inactive due to the presence of a point mutation T-a-G at the second base of exon 9. In a specific embodiment, a pig animal is provided, in the one that both alleles of the gene of the alpha-1,3-GT are inactivated, by which an allele is inactivated by a construction of modification directed to Exon 9 (see, for example, Figure 6) and the other allele is inactive due to the presence of a mutation punctual T-a-G at second base of exon 9 (Figure 2). The directed modification, for example, You can also go to exon 9, and / or exons 4-8.

In a further embodiment, an allele is inactive by a directed genetic modification event and the other allele is inactive due to the presence of a mutation punctual T-a-G at the second base of the exon 9 of the alpha-1,3-GT gene. In a specific embodiment, an allele is inactivated by a modification construction directed to Exon 9 (see, for example, Figure 6) and the other allele is inactive due to the presence of a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene. A method  To clone these pigs includes: enucleating a non-human oocyte, fuse the oocyte with a donor nucleus of a swine cell that lacks functional alpha1,3GT expression, and implant the embryo obtained by nuclear transfer in a non-human mother substitute In another aspect, the invention provides a method for produce a non-human animal deficient in alpha-1,3-GT comprising the stages listed in claim 24.

A method to produce viable pigs that lack any expression of functional alpha-1,3-GT may include cross a heterozygous male pig for the gene of the alpha-1,3-GT with a female pig heterozygous for the gene of the alpha-1,3-GT. Pigs can be heterozygous due to the genetic modification of an allele of the alpha-1,3-GT gene to avoid expression of that allele. Pigs can be heterozygous due to the presence of a point mutation in an allele of the gene of the alpha-1,3-GT. Punctual mutation it can be a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene. A method to produce a pig animal that lacks any expression of functional alpha-1,3-GT can include crossing a male pig that contains a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene with a female pig that contains a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene, or vice versa.

In another aspect of the present invention, a method is provided for selecting cells that lack galactose alpha-1,3 galactose expression, as reported in claim 17. The screening method can be used to determine whether porcine cells express galactose. alpha-1,3 galactose on the cell surface. The bacterial toxin, toxin A produced by Clostridium difficile , is used to select cells that lack galactose alpha-1,3-galactose expression.

Exposure to C. difficile toxin can cause rounding of cells that show this epitope on its surface, releasing the cells from the plaque matrix. Both gene deletions by directed modification and mutations that disable the function or expression of the enzyme can be detected using this method of selection. Cells that lack expression on the cell surface of galactose alpha 1,3-galactose, identified using the described toxin-mediated selection described, or produced using conventional methods of gene inactivation including targeted gene modification, can then be used to produce pigs that lack the expression of functional alpha1,3GT.

Other embodiments of the present invention they will be apparent to those skilled in the art in light of the following description of the invention, the claims and what It is known in the art.

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Brief description of the drawings

Figure 1 is a graph depicting the relative lytic effects of complement on fetus cells.
680B1-4.

Figure 2 represents a short segment of the coding region of the alpha-1,3-GT gene (see GenBank Accession No. L36152) in which there is the point mutation selected by toxin A. The upper sequence exists in the wild type; the lower sequence shows the change due to the point mutation in the second
allele

Figure 3 is a representation of a model three-dimensional UDP binding site of bovine alpha1.3GT. He aromatic ring of the rest of tyrosine (foreground, white) can observed very close to the UDP uracil base (scale of gray)

Figure 4 is a photograph of cloned pigs deficient in alpha-1,3-GT homozygous produced by the methods of the invention, born on July 25, 2002.

Figure 5 is a graph depicting levels of anti-alpha-1,3-gal IgM before and after injections of islet-like cell groups (ICC) of piglets in alpha-1,3-GT KO mice. Each mouse received three serial injections of ICC via ip (200-500 ICC per injection) for 4 days. The three wild-type piglet (WT) ICC receptors showed a significant elevation of 1.3 gal anti-alpha IgM titer and subsequent return to the initial measurement 4 weeks after ICC implants. The sera of the three mice injected with ICC of piglet alpha-1,3-GT DKO maintained low baseline titers of anti-alpha-1,3-gal IgM during the 35-day observation time (Phelps et al ., Science 299: 411-414, 2003, Figure S4).

Figure 6 is a locus diagram porcine alpha-1,3-GT, corresponding to the genomic sequences of alpha-1,3-GT that can be used as 5 'and 3' arms in knockout vectors of alpha-1,3-GT, and the structure of locus modified in a directed way after recombination homologous The names and positions of the primers used to 3 'PCR and long range PCR are indicated by short arrows. The short bar indicates the probe used for the analysis of Southern transfer of alpha-1,3-GT. The predicted size of Southern bands with digestion with BstEII for both the alpha-1,3-GT locus endogenous as for the locus alpha-1,3-GT modified form directed.

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Detailed description of the invention

It has now been shown that a viable pig animal that lacks any expression of alpha 1,3 functional galactosyltransferase. The present invention provides complete inactivation of both alleles of the alpha 1,3 gene galactosyltransferase in pigs, thus overcoming this ancient obstacle and making xenotransplantation a reality. Remove the expression of this gene, which causes the absence of galactose alpha 1,3-galactose on the cell surface, represents the first and main stage in the elimination of hyperacute rejection in xenotransplant therapy pig-to-human being. The invention also provides organs, tissues, and cells obtained from said pig animal, which are useful for xenotransplantation.

In one aspect, the invention provides organs, tissues and / or purified cells or cell lines or substantially  pure pigs obtained from pigs that lack any functional alpha-1,3-GT expression where both alleles of the gene of the alpha-1,3-GT are inactive, and where an allele becomes inactive through at least one mutation punctual. Organs, tissues, cells or cell lines can be useful for xenotransplantation.

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Definitions

As used in this document, it is understood that the term "animal" (as in "modified (or altered) animal genetically ") includes any non-human animal, particularly any non-human mammal, including, although without limitation, pigs, sheep, goats, cattle (cattle), deer, mules, horses, monkeys, dogs, cats, rats, mice, Birds, chickens, reptiles, fish, and insects. In an embodiment of the invention, genetically altered pigs are provided and production methods thereof.

As used in this document, an "organ" it is an organized structure, which can be composed of one or more fabrics An "organ" performs one or more biological functions specific. The organs include, without limitation, the heart, the liver, kidney, pancreas, lung, thyroid, and skin.

As used herein, a "tissue" it is an organized structure that comprises cells and substances intracellular surrounding them. The "tissue", alone or together with other cells or tissues can perform one or more functions Biological

As used in this document, the terms "pig", "pig animal", "pig" and "pig" they are generic terms that refer to the same type of animal regardless of gender, size, or lineage.

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I. Directed Genetic Modification of the gene of the alpha-1,3-GT

The invention provides a pig as mentioned in claim 1.

In an embodiment of the present invention, they provide pig animals in which both alleles of the gene of the alpha-1,3-GT are inactive, and in those that an allele becomes inactive through at least one point mutation and in which an allele of the gene of the alpha-1,3-GT is inactivated by a directed genetic modification event. In other embodiment of the present invention, both alleles of the gene of the alpha-1,3-GT is inactivated by an event of directed genetic modification. In a embodiment, the gene can be modified in a directed manner by homologous recombination. In other embodiments, the gene may alter, that is, a part of the genetic code can be altered, thus affecting the transcription and / or translation of that segment of the gene. For example, the alteration of a gene can happen through substitution techniques, deletion ("knockout") or insertion ("knockin"). Additional genes can be inserted to a desired protein or regulatory sequence that modulates the transcription of an existing sequence.

In embodiments of the present invention, the alleles of the alpha-1,3-GT gene are they become inactive, so that the enzyme resulting alpha-1,3-GT can no longer generate galactose alpha1, 3-galactose on the cell surface In one embodiment, the gene of the alpha-1,3-GT can be transcribed in RNA, but cannot be translated into protein. In another embodiment, the alpha-1,3-GT gene can Transcribe in a truncated form. Said truncated RNA may not translate or can be translated into a non-functional protein. In a alternative embodiment, the gene of the alpha-1,3-GT can be inactivated in such so that gene transcription does not happen. In one embodiment additional, the alpha-1,3-GT gene can be transcribed and then translated into a protein not functional.

Pigs that have two inactive alleles of the alpha-1,3-GT gene are not from natural origin It was surprisingly discovered that while tried to eliminate the second allele of the gene from the alpha-1,3-GT through a directed genetic modification event, a point mutation, which prevented the second allele from producing functional alpha1,3GT.

An allele of the gene of the alpha-1,3-GT becomes inactive at through at least one point mutation. In another embodiment, both alleles of the alpha-1,3-GT gene they can become inactive, through at least one mutation punctual. In one embodiment, this point mutation can happen through an event of directed genetic modification. In Another embodiment, this point mutation can be of natural origin. In a further embodiment, mutations in the gene can be induced. of the alpha-1,3-GT using an agent mutagenic

In a specific embodiment, the mutation punctual can be a T-a-G mutation at the second base of exon 9 of the gene of the alpha-1,3-GT (Figure 2). Pigs that carry a point mutation of natural origin in the gene of the alpha-1,3-GT allow production of pigs deficient in alpha1,3GT free of resistance genes to antibiotics and therefore have the potential to create a product Safer for human use. In other embodiments, they may exist. at least two, at least three, at least four, at least five, at minus ten or at least twenty point mutations to return to inactive alpha-1,3-GT gene. In other embodiments, pigs are provided in which both alleles of the alpha-1,3-GT gene contain point mutations that avoid any expression of alpha1,3GT functional. In a specific embodiment, pigs are provided that contain the T-a-G mutation in the second base of exon 9 in both alleles of the gene of the alpha-1,3-GT (Figure 2).

Another embodiment of the present invention provides a pig animal, in which both alleles of the gene of the alpha-1,3-GT are inactivated, so which one allele is inactivated by a modification event directed genetics and the other allele is inactivated by a mutation punctual. In one embodiment, a pig animal is provided, in the one that both alleles of the gene of the alpha-1,3-GT are inactivated, so which one allele is inactivated by a modification event directed genetics and the other allele is inactive due to the presence of a point mutation T-a-G in the second base of exon 9. In a specific embodiment, provides a pig animal, in which both alleles of the gene of the alpha-1,3-GT are inactivated, so which one allele is inactivated by a modification construct directed to Exon 9 (see, for example, Figure 6) and the other allele becomes inactive due to the presence of a point mutation T-a-G at the second base of the exon 9.

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Swine cell types

Swine cells that can be modified genetically can be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disintegrated preparation of an embryo, fetus or complete adult animal or a part of it. For example, swine cells can be selected from the group consisting of, but not limited to, epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, heart muscle cells, other muscle cells, cells of the granulose, cumulus cells, epidermal cells, cells endothelial, islet cells of Langerhans, cells blood, blood precursor cells, bone cells, cells bone precursors, neuronal stem cells, stem cells primordial, hepatocytes, keratinocytes, endothelial cells of umbilical vein, aortic endothelial cells, cells Endothelial microvascular, fibroblasts, crashed cells liver, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, cells Schwann, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes; monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, cells of the pituitary, adrenal cells, hair cells, cells of the bladder, kidney cells, retinal cells, retinal canes, retinal cones, heart cells, pacemaker cells, splenic cells, antigen presenting cells, cells memory, T cells, B cells, plasma cells, cells muscle, ovarian cells, uterus cells, cells of the prostate, vaginal epithelial cells, sperm cells, testicular cells, germ cells, ovules, cells leydig, peritubular cells, sertoli cells, cells luteal, cervical cells, endometrial cells, cells mammary, follicular cells, mucous cells, hair cells, non keratinized epithelial cells, epithelial cells keratinized, lung cells, calciform cells, cells columnar epithelial, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts.

Non-human embryonic stem cells can be used. An embryonic stem cell line can be used or recently non-human embryonic stem cells can be obtained from a host, such as a pig animal. Cells can be cultured in a fibroblast feed layer or cultured in the presence of leukemia inhibitor factor (LIF). In a preferred embodiment, the porcine cells may be fibroblasts; In a specific embodiment, the porcine cells may be fetal fibroblasts. Fibroblastic cells are a preferred type of somatic cell because they can be obtained from developing fetuses and adult animals in large quantities. These cells can easily propagate in vitro with a rapid doubling time and can be clonally propagated for use in targeted gene modification procedures.

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Directed modification constructions Homologous Recombination

Homologous recombination allows modifications site-specific in endogenous genes and therefore can Design new alterations in the genome. In recombination homologous, the incoming DNA interacts with and integrates into a site in the genome that contains a DNA sequence substantially homologous In non-homologous integration ("random" or "illegal"), the incoming DNA is not in a sequence homologous in the genome but it integrates anywhere, in One of the large number of potential places. In general, studies  with higher eukaryotic cells have revealed that the frequency of homologous recombination is much less than the frequency of random integration The proportion of these frequencies has direct implications for "targeted gene modification" which depends on integration by homologous recombination (it is that is, recombination between the "directed modification DNA" exogenous and the corresponding "target DNA" in the genome).

Several articles describe the use of homologous recombination in mammalian cells. Illustrative of these articles are Kucherlapati et al ., Proc. Natl Acad. Sci. USA 81: 3153-3157, 1984; Kucherlapati et al ., Mol. Cell Bio. 5: 714-720, 1985; Smithies et al ., Nature 317: 230-234, 1985; Wake et al ., Mol. Cell Bio. 8: 2080-2089, 1985; Ayares et al ., Genetics 111: 375-388, 1985; Ayares et al ., Mol. Cell Bio. 7: 1656-1662, 1986; Song et al ., Proc. Natl Acad. Sci. USA 84: 6820-6824, 1987; Thomas et al . Cell 44: 419-428, 1986; Thomas and Capecchi, Cell 51: 503-512, 1987; Nandi et al ., Proc. Natl Acad. Sci. USA 85: 3845-3849, 1988; and Mansour et al ., Nature 336: 348-352, 1988. Evans and Kaufman, Nature 294: 146-154, 1981; Doetschman et al ., Nature 330: 576-578, 1987; Thomas and Capecchi, Cell 51: 503-512, 1987; Thompson et al ., Cell 56: 316-321, 1989.

The present invention uses recombination homologous to inactivate the gene of the alpha-1,3-GT in cells, such as the porcine cells described above. DNA can comprise at least a part of the gene (s) in the particular locus with the introduction of an alteration in the minus one, optionally both copies, of the native gene (s), to avoid expressing functional alpha1,3GT. The alteration can be an insertion, deletion, replacement or combination thereof. When the alteration is introduced in only one copy of the gene being by inactivating, cells that have a single unmutated copy of the target gene are amplified and can undergo a second stage of directed modification, where the alteration may be the same or different from the first alteration, usually different, and where a deletion, or replacement, that may be overlapping is involved with at least part of the alteration originally introduced. In this second stage of directed modification, a directed modification vector with the same homology arms, but that contains selection markers in different mammals. The resulting transformants are screened for the absence of a functional target antigen and cell DNA can be scanned additionally to ensure the absence of a target gene type wild. Alternatively, homozygosity can be achieved in as for a phenotype by crossing heterozygous hosts for the mutation

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Targeted modification vectors

The modification of a target locus of a cell can occur by introducing DNA into the cells, where the DNA has homology to the target locus and includes a marker gene, which allows the selection of cells comprising the integrated construct. Homologous DNA in the target vector will recombine with the chromosomal DNA in the target locus. The marker gene can be flanked on both sides by homologous DNA sequences, a 3 'recombination arm and a 5' recombination arm. Methods for constructing directed modification vectors have been described in the art, see, for example, Dai et al ., Nature Biotechnology 20: 251-255, 2002; WO 00/51424, Figure 6.

Various constructs can be prepared for homologous recombination in a target locus. The construction may include at least 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp homologous sequence with the target locus. The sequence may include any contiguous sequence of the porcine gene of alpha-1,3-GT (see, for example, GenBank Accession No. L36152, WO 01/30992 of the University of Pittsburgh of the Commonwealth System of Higher Education ; Alexion WO 01/123541,
Inc.).

Various considerations may be involved in determining the degree of homology of target DNA sequences, such as, for example, the size of the target locus, the availability of sequences, the relative efficacy of double cross-linking events in the target locus and similarity. of the target sequence with others
sequences

Targeted modification DNA may include a sequence in which substantially isogenic DNA flanks the desired sequence modifications with a target sequence corresponding in the genome to modify. Sequence substantially isogenic can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the target sequence corresponding (except for sequence modifications desired). Targeted modification DNA and target DNA preferably they can share stretches of DNA of at least approximately 75, 150 or 500 base pairs that are 100% identical. Accordingly, the directed modification DNA can be obtained of cells closely related to the cell line being modifying in a directed way; or the directed modification DNA can be obtained from cells of the same cell or animal line that the cells that are being modified in a directed way.

DNA constructs can be designed to modify the endogenous target alpha1,3GT. The homologous sequence for directing the construction can have one or more deletions, insertions, substitutions or combinations thereof. The alteration may be the insertion of a marker selection gene merged in reading phase with the sequence upstream of the gene Diana.

Suitable selection marker genes include, but are not limited to: genes that confer the ability to grow on certain media substrates, such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine phosphoribosyltransferase) that confers the ability to grow in medium HAT (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene (guanine / xanthine phosphoribosyltransferase) that allows growth in MAX medium (mycophenolic acid, adenine, and xanthine). See, for example, Song, KY., Et al . Proc. Nat'l Acad. Sci. USA 84: 6820-6824 (1987); Sambrook, J., et al ., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), Chapter 16. Other examples of selection markers include: genes that confer resistance to compounds such as antibiotics , genes that confer the ability to grow on selected substrates, genes that encode proteins that produce detectable signals such as luminescence, such as fluorescent green protein, enhanced fluorescent green protein (eGFP). A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin (neo) resistance gene (Southern, P., and P. Berg, J. Mol. Appl. Genet. 1: 327-341 (1982)); and the hygromycin (hyg) resistance gene (Nucleic Acids Research 11: 6895-6911 (1983), and Te Riele, H., et al ., Nature 348: 649-651 (1990)). Other selection marker genes include: acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP) , yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selection markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and
tetracycline

Methods for incorporating antibiotic resistance genes and negative selection factors will be known to those skilled in the art (see, for example, WO 99/15650; US Patent No. 6,080,576; United States No. 6,136,566; Niwa et al ., J. Biochem. 113: 343-349 (1993); and Yoshida et al ., Transgenic Research 4: 277-287 (1995)).

TABLE 1 Selection marker genes that emit signals detectable

2

3

4

5

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Combinations of selection markers. For example, to dial alpha1.3GT, you can clone a neo gene (with or without its own promoter, as has been discussed above) in a DNA sequence that is homologous to alpha-1,3-GT gene. To use a combination of markers, the gene can be cloned HSV-tk so that it is outside the DNA of directed modification (another selection marker could be placed on the opposite flank, if desired). After entering the construction of DNA in the cells to be modified in a directed way, the cells can be selected in the appropriate antibiotics. In this particular example, those cells that are resistant to G418 and ganciclovir are more likely to have arisen from homologous recombination in which the neo gene has been combined in the alpha-1,3-GT gene but it has lost the tk gene because it was located outside the region of double crosslinking.

Deletions can be at least about 50 bp, more usually about 100 bp, and generally no more than about 20 kbp, where the deletion can usually include at least a part of the region encoder that includes a part of one or more exons, a part of one or more introns, and may or may not include a part of the regions non-flanking encoders, particularly the non-region 5 'coding (transcription regulatory region). So, the homologous region can extend beyond the region coding in the 5 'non-coding region or as an alternative in the 3 'non-coding region. Insertions generally cannot exceed 10 kbp, usually do not exceed 5 kbp, being generally at least 50 bp, more usually at least 200 pb.

The homology region (s) may (n) include mutations, where mutations can further inactivate the target gene, providing a phase shift, or changing a key amino acid, or the mutation can correct a dysfunctional allele, etc. Mutation It may be a subtle change, which does not exceed approximately 5% of homologous flanking sequences. When the mutation of a gene, the marker gene can be inserted into an intron or an exon.

The construction can be prepared according to methods known in the art, various fragments can be joined, introduced into appropriate vectors, cloned, analyzed and then further manipulated until the desired construction has been achieved. Various modifications to the sequence can be made, to allow restriction analysis, excision, probe identification, etc. Silent mutations can be introduced, as desired. In various phases, restriction analysis, sequencing, amplification with the polymerase chain reaction, primer repair, in vitro mutagenesis, etc. can be used.

The construct can be prepared using a bacterial vector, which includes a prokaryotic replication system, for example, an origin recognizable by E. coli , at each stage the construct can be cloned and analyzed. A marker can be used, the same or different from the marker to be used for insertion, which can be removed before introduction into the target cell. Once the vector containing the construction has been completed, it can be further manipulated, such as by deletion of the bacterial sequences, linearization, introducing a short deletion into the homologous sequence. After the final manipulation, the construction can be introduced into the cell.

The present invention further includes recombinant constructs containing sequences of the alpha-1,3-GT gene. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in direct or inverse orientation. The construct may also include regulatory sequences including, for example, a promoter, functionally linked to the sequence. Those skilled in the art know large amounts of suitable vectors and promoters, and are commercially available. The following vectors are provided by way of example. Bacterial: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotes: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPv, pMSG, pSVL (Pharmacia), viral origin vectors (M13 vectors, bacterial phage 1 vectors, adenoviral vectors, and retroviral vectors), high, low and adjustable copy number, vectors that have compatible replicons for use in combination in a single host (pACYCI 84 and pBR322) and eukaryotic episomic replication vectors (pCDM8). Other vectors include prokaryotic expression vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Corp.), pGEMEX-1, and pGEMEX-2 ( Promega, Inc.), the vectors pET (Novagen, Inc.), pTrc99A, pKK223-3, the vectors pGEX, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2 and pKK388-1 (Clontech, Inc .), and pProEx-HT (Invitrogen, Corp.) and variants and derivatives thereof. Other vectors include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneoVS (ptech3) , pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B , and C, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3, pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) and variants or derivatives thereof. Additional vectors that can be used include: pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagomids, YAC (artificial yeast chromosomes), BAC (bacterial artificial chromosomes), P1 ( Escherichia coli phage), pQE70, pQE60, pQE9 (quagan) , pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR533-3, pDR5 pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Invitrogen), pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His, pcDNA3.1 / His,
Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZ, pGAPZ, pGAPZ, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, p1, p2, p2, p2, p2, p2, pR1, p2, pRD, p2, p2, p2, pRD, p2, pR1, p2, pRD1, p2, p2, pRD1, p2, p1, pR1 , pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1 / Amp, pcDNA3.1, pcDNA3.1 / Zeo, pSe, SV2, pRc / CMV2, pRc / RSV, pREP4, pREP7, pREP8, pREP9, pREP10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac of Invitrogen; ExCell, gt11, pTrc99A, pKK223-3, pGEX-1 T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1 , pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia; pSCREEN-1b (+), pT7Blue (R), pT7Blue-2, pCITE-4abc (+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC, pT7Blue-2, SCREEN-1, BIueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b (+), pET-21abcd (+), pET-22b (+), pET-23abcd (+), pET-24abcd (+), pET-25b (+), pET-26b (+), pET -27b (+), pET-28abc (+), pET-29abc (+), pET-30abc (+), pET-31b (+), pET-32abc (+), pET-33b (+), pBAC- 1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp, pBACsurf-1, plg, Signal plg, pYX, Select Vecta-Neo, Select Vecta-Hyg, and Select Vecta-Gpt from Novagen ; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis pSEAP2-Basic, pSEAP2-Control, pSEAP2-Promoter, pSEAP2-Enhancer, p gal-Basic, p gal-Control, p gal-Promoter, p gal-Enhancer, pCMV, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3, pYEX-S1, pBacPac-His8 9, pAcUW31, BacPAK6, pTriplEx, gt10, gt11, pWE15, and TriplEx from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS +/-, pBluescript II SK +/-, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS +/-, pBC KS +/-, pBC SK +/-, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI / MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, GOG45, PNE45, GOG45, GOG45, GOG45, GOG45, GOG45, PNE45, GAL45, GAL45 , pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and Stratagene pRS416 and variants or derivatives thereof. Inverse double hybrid and double hybrid vectors can also be used, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof. Any other plasmid and vector can be used as long as it is replicable and viable in the host.

Techniques that can be used to allow DNA construction to enter the host cell include co-precipitation of calcium phosphate / DNA, microinjection of DNA in the nucleus, electroporation, fusion of the bacterial protoplast with intact cells, transfection, or any other technique known to The specialists in the art. The DNA can be single-stranded or double-stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transfecting mammalian cells see, for example, Keown et al ., Methods in Enzymology Vol. 185, p. 527-537 (1990).

In a specific embodiment, heterozygous knockout cells can be produced by transfection of primary porcine fetal fibroblasts with a knockout vector containing the alpha-1,3-GT sequence isolated from isogenic DNA. As described in Dai et al . (Nature Biotechnology, 20: 451-455), the 5 'arm may be 4.9 kb and be composed of a large fragment of intron 8 and the 5' end of exon 9. The 3 'arm may be and be composed by exon 9 sequence. The vector may incorporate a promoter capture strategy, using, for example, IRES (internal ribosome entry site) to initiate the translation of the Neo gene (see, for example, Figure 6).

Homologously recombined cell selection

The cells can then be grown in medium appropriately selected to identify the cells that They provide the appropriate integration. The absence of the marker gene of selection inserted in the gene of the alpha-1,3-GT establishes integration of the target construct in the host genome. Those cells which show the desired phenotype can then be analyzed additionally by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, etc. for analyze the DNA to establish if recombination has happened homologous or non-homologous. This can be determined using probes for the insert and then sequencing the 5 'and 3' regions that flank the insert for the presence of the gene of the alpha-1,3-GT that extends more beyond the flanking regions of the construction or identifying the presence of a deletion, when said deletion is introduced. Primers can also be used that are complementary to a sequence within the construction and complementary to a sequence outside the construction and in the target locus. Of this mode, only DNA duplexes that have both can be obtained primers present in the complementary chains if it has happened homologous recombination. Demonstrating the presence of the sequences primers or the expected size sequence, existence is supported of homologous recombination.

The polymerase chain reaction used to explore homologous recombination events is known in the art, see, for example, Kim and Smithies, Nucleic Acids Res. 16: 8887-8903, 1988; and Joyner et al ., Nature 338: 153-156, 1989. The specific combination of a mutant polyoma enhancer and a thymidine kinase promoter to direct the neomycin gene has been shown to be active in both embryonic stem cells and cells. EC by Thomas and Capecchi, supra , 1987; Nicholas and Berg (1983) in Teratocarcinoma Stem Cell, eds. Siver, Martin and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, NY (p. 469-497); and Linney and Donerly, Cell 35: 693-699, 1983.

The cell lines obtained from the first directed modification round are probably heterozygous for the allele modified in a directed way. The homozygosity, in the that both alleles are modified, can be obtained from several modes. One approach is to grow several cells in which it has been modified a copy and then subject these cells to another round of directed modification using a different selection marker. As an alternative, homozygotes can be obtained by crossing animals heterozygous for the modified allele, according to the Traditional Mendelian genetics. In some situations, it can be desirable to obtain two different modified alleles. This can be achieved by successive rounds of targeted gene modification or crossing heterozygotes, each of which carries one of the Desired modified alleles.

Mutation induced in the alpha 1,3 GT locus

In certain different embodiments, the methods of the invention involve the intentional introduction of a mutation by a mutagenic agent. Examples of agents mutagenic known in the art and suitable for use in the The present invention includes, but is not limited to, mutagens. chemicals (for example, chemical agents that interleave in the DNA or that bind to DNA such as N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), mustard gas, ICR191 and the like; see, for example, E. C. Friedberg, G. C. Walker, W. Siede, DNA Repair and Mutagenesis, ASM Press, Washington DC (1995), mutagens physical (for example, UV radiation, radiation, x-rays), mutagens biochemicals (e.g. restriction enzymes, mutagens of DNA repair, DNA repair inhibitors, and DNA error-prone polymerases and replication proteins), as well as Transposon insertion. According to the methods of the In the present invention, cells in culture can be exposed to one of these agents, and any mutation that can be selected cause galactose removal alpha1,3-galactose on the cell surface, by example, by exposure to toxin A.

Preferred doses of chemical mutagens to induce cell mutations are known in the art, or can be readily determined by those skilled in the art using mutagenesis assays known in the art. Chemical mutagenesis of cells in vitro can be achieved by treating the cells with various doses of the mutagenic agent and / or controlling the time of exposure to the agent. By assessing the exposure to the mutagenic agent and / or the dose, it is possible to perform the optimum degree of mutagenesis for the intended purpose, thereby mutating a desired amount of genes in each target cell. For example, useful doses of ENU may be 0.1-0.4 mg / ml for approximately 1-2 hours. In another example, useful doses of EMS may be 0.1-1 mg / ml for approximately 10-30 hours. In addition, lower and higher doses and exposure times can also be used to achieve the desired mutation frequency.

II. Identification of Non-Expressing Cells Alfa-1,3-GT Functional

In another aspect of the present invention, provides a selection method to determine if cells pigs lack the expression of functional alpha-1,3-GT.

The bacterial toxin, toxin A produced by Clostridium difficile , is used to select cells that lack the galactose alpha1,3-galactose cell surface epitope. Exposure to C. difficile toxin can cause rounding of cells that show this epitope on its surface, releasing the cells from the plaque matrix. Both targeted gene knockouts and mutations that disable function or enzymatic expression can be detected using this method of selection. Cells that lack expression on the cell surface of the galactose alpha1,3-galactose epitope, identified using the toxin-mediated selection A described, or produced using conventional methods of gene inactivation including targeted gene modification, can then be used to produce pigs, in which both alleles of the alpha 1,3 GT gene are inactive.

In one embodiment, the selection method can detect the elimination of the alpha 1,3GT epitope directly, since either due to targeted elimination of the 1.3GT alpha gene by homologous recombination, or a mutation in the gene that causes a enzyme that does not work or that is not expressed. The selection by Antibiotic resistance has been used more commonly for exploration (see above). This method can detect the presence of the resistance gene in the modification vector directed, but does not directly indicate if the integration was a directed recombination event or an integration random Certain technology, such as capture technology poly A and promoter, increases the probability of events directed, but again, does not give direct evidence that it has achieved the desired phenotype, a cell deficient in epitopes 1.3 gal gal gal on the cell surface. In addition, they can be used negative forms of selection to select integration directed; in these cases, the gene of a lethal factor is inserted to the cells in such a way that only targeted events allow the cell to avoid death. Selected cells by these methods they can then be tested for gene alteration, vector integration and finally elimination of the alpha epitope 1.3 gal. In these cases, as the selection is based on detection of the integration of the directed modification vector and not in the altered phenotype, only targeted knockouts can be detected, no point mutations, gene rearrangements or truncation or other such modifications.

Toxin A, a cytotoxin produced by the Clostridium difficile bacteria, specifically binds to the terminal carbohydrate sequence of galactose alpha1,3-galactose gal alpha 1-3 gal beta 1-4GlcNAc. Binding to this receptor mediates a cytotoxic effect in the cell, causing it to change morphology and, in some cases, be released from the matrix of the plaque. Under controlled conditions, cells that do not carry this marker are not affected by the toxin. Therefore, in one embodiment, in order to determine whether or not the 1,3 gal alpha epitope has been successfully removed by targeted elimination or gene mutation of the alpha-1,3-GT gal locus, cells that do not carry the epitope can be selected. Exposure to toxin A can be toxic to cells that carry the epitope, and promotes the selection of those cells in which the gene has been successfully inactivated.

Cells useful as nuclear donors for the production of genetically altered animals (e.g. pigs) that have the locus gal alpha 1.3 removed or mutated can be selected by exposure of the cells to C. difficile toxin A.

Toxin A, one of the two cytotoxins produced by Clostridium difficile , has high binding affinity for the galactose sequence alpha1,3-galactose gal alpha 1,3-gal beta 1,4GlcNAc found on the surface of a variety of cell types (Clark et al ., Arch. Biochem. Biophys. 257 (1): 217-229, 1987). This carbohydrate seems to serve as a functional receptor for toxin A, since cells that present this epitope on its surface are more sensitive to the cytotoxic effect of toxin A than cells lacking this receptor. Sensitive cells exposed to toxin A in culture show cell rounding, probably due to depolymerization of actin and the resulting changes in the integrity of the cytoskeleton (Kushnaryov et al ., J. Biol. Chem. 263: 17755-17762 (1988) and Just et al ., J. Clin. Invest. 95: 1026-1031, 1995). These cells can be selectively removed from the culture, as they rise from the matrix and float in suspension, leaving the unaffected cells firmly attached to the surface of the plate.

Exposure of cells to toxin A. In a embodiment, the bound cells are exposed to toxin A as a component of the cell culture medium. After a fixed time of exposure, the medium containing toxin A and the toxin A sensitive cells released, the plate is washed, and replenishes the medium, without toxin A. The exposure to toxin A is repeated over a period of days to remove sensitive cells to toxin bound plaques, and allow insensitive cells proliferate and expand. The purified toxin A can be used in the methods of the present invention (commercially available, see, for example, Techlab Inc., Cat. No. T3001, Blacksburg, VA). Untreated unrefined toxin A can also be used (available on the market, see, for example, Techlab Inc. Cat. No. T5000 or T3000, Blacksburg, VA), which may require an initial degree to determine the effective dosage for selection.

Serum based selection method

The screening procedure can be performed using serum containing complement factors and natural antibodies against the epitope gal alpha 1.3 gal (see, for example, Koike et al ., Xenotransplantation 4: 147-153, 1997). Serum exposure of a human or non-human primate that contains anti-Gal antibodies may cause cell lysis due to specific binding of the antibody and complement activation in cells showing the 1.3 gal gal alpha epitope. Therefore, alpha-1,3-GT deficient cells will remain alive and therefore can be selected.

Additional characterization of porcine cells that lack the 1.3GT functional alpha expression

Swine cells that are believed to lack alpha-1,3-GT expression Functional can be further characterized. Bliss characterization can be done by the following techniques, including, but not limited to: PCR analysis, analysis of Southern blot, Northern blot analysis, lectin specific binding assays, and / or analysis of sequencing

PCR analysis can be used as described in the art (see, for example, Dai et al . Nature Biotechnology 20: 431-455) to determine the integration of targeted modification vectors. Amplimers can originate in the antibiotic resistance gene and extend into a region outside the vector sequence. Southern analysis (see, for example, Dai et al . Nature Biotechnology 20: 431-455) can also be used to characterize global modifications in the locus, such as the integration of a directed modification vector into the 1.3GT alpha locus. Although Northern analysis can be used to characterize the transcript produced from each of the alleles.

Specific lectin binding, using lectin GSL IB4 of Griffonia (Bandeiraea) simplicifolia (Vector Labs), a lectin that specifically binds to the rest of carbohydrate gal alpha 1.3 gal, and FACS binding analysis (cell separation activated by fluorescence) can determine whether the epitope alpha 1,3 Gal is present or not in the cells. This type of analysis involves the addition of GSL-IB4 lectin labeled with fluorescein to cells and subsequent cell separation.

In addition, analysis of cDNA sequencing produced from the RNA transcript for  determine the precise location of any mutation in the allele of the alpha 1.3GT.

III. Swine Animal Production

In yet another aspect, the present invention provides a method to produce viable pigs in which both alleles of the alpha-1,3-GT gene are they have become inactive, where the method is as indicated in the claim 24. In one embodiment, the pigs are produced by cloning using a donor nucleus of a swine cell in which both alleles of the gene of the alpha-1,3-GT have been inactivated. In one embodiment, both alleles of the gene of the alpha-1,3-GT is inactivated by a directed genetic modification event. In other embodiment, both alleles of the gene of the alpha-1,3-GT are inactivated due to the presence of a point mutation. In another embodiment, an allele is inactive by a directed genetic modification event and the other allele is inactivated by a point mutation. In a further embodiment, an allele is inactivated by an event of directed genetic modification and the other allele is inactivated due to to the presence of a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene. In a specific embodiment, an allele is inactivated by a modification construction directed to Exon 9 (Figure 6) and the other allele becomes inactive due to the presence of a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene. In other embodiment, a method for cloning said pigs includes: enuclear an oocyte, fuse the oocyte with a donor nucleus of a cell swine in which both alleles of the gene of the alpha-1,3-GT have been inactivated, and implant the embryo obtained by nuclear transfer in a mother substitute

Alternatively, a method for produce viable pigs that lack any expression of alpha-1,3-GT inactivating both alleles of the alpha-1,3-GT gene in cells non-human embryonic mother, which can then be used to produce offspring

Genetically altered nonhuman animals that They can be created by modifying zygotes directly. For mammals, the modified zygotes can then be introduced into the uterus of a non-human pseudo-pregnant female capable of carrying The term animal. For example, if nonhuman animals are desired complete that lack the gene of the alpha-1,3-GT, then they can be modified in a targeted way embryonic stem cells obtained of this animal and then introduced into blastocysts so that the Modified cells grow in chimeric animals. For cells embryonic stem, a stem cell line can be used non-human embryonic or newly non-human stem cells obtained.

Totipotent cells can be non-human embryonic stem (ES) cells. The isolation of ES cells from blastocysts, the establishment of ES cell lines and their subsequent culture are performed by conventional methods described, for example, by Doetchmann et al ., J. Embryol. Exp. Morph. 87: 27-45 (1985); Li et al ., Cell 69: 915-926 (1992); Robertson, EJ "Tetracarcinomas and Embryonic Stem Cells: A Practical Approach", ed. EJ Robertson, IRL Press, Oxford, England (1987); Wurst and Joyner, "Gene Targeting: A Practical Approach", ed. AL Joyner, IRL Press, Oxford, England (1993); Hogen et al ., "Manipulating the Mouse Embryo: A Laboratory Manual", eds. Hogan, Beddington, Costantini and Lacy, Cold Spring Harbor Laboratory Press, New York (1994); and Wang et al ., Nature 336: 741-744 (1992). Totipotent cells can be non-human embryonic (EG) germ cells. Embryonic germ cells are undifferentiated cells functionally equivalent to ES cells, that is, they can be grown and transfected in vitro , then contribute to the somatic and germ cell lines of a chimera (Stewart et al ., Dev. Biol. 161: 626 -628 (1994)). EG cells are obtained by culturing primary germ cells, gamete progenitors, with a combination of growth factors: leukemia inhibitor factor, steel factor and basic fibroblast growth factor (Matsui et al ., Cell 70: 841 -847 (1992); Resnick et al ., Nature 359: 550-551 (1992)). The culture of EG cells can be performed using methods described in the article by Donovan et al ., "Transgenic Animals, Generation and Use", Ed. L M. Houdebine, Harwood Academic Publishers (1997), and in the original bibliography mentioned in this document.

Non-human tetraploid blastocysts can be obtained for use in the invention by natural production and development of zygotes, or by methods known by electrofusion of two cell embryos and can be subsequently cultured as described, for example, by James et al ., Genet. Res. Change 60: 185-194 (1992); Nagy and Rossant, "Gene Targeting: A Practical Approach", ed. AL Joyner, IRL Press, Oxford, England (1993); or by Kubiak and Tarkowski, Exp. Cell Res. 157: 561-566 (1985).

The introduction of non-human ES cells or non-human EG cells in the blastocysts can be performed by any method known in the art. A suitable method for the purposes of the present invention is the microinjection method described by Wang et al ., EMBO J. 10: 2437-2450 (1991).

As an alternative, using stem cells modified non-human embryonic animals can be produced transgenic Modified Embryonic Stem Cells genetically they can be injected into a blastocyst and then taken  term in a female host mammal according to techniques conventional. The heterozygous progeny can then be explored for the presence of the alteration at the site of the target locus, using techniques such as PCR or Southern blotting. After of mating with a wild-type host of the same species, the resulting chimeric progeny can then mate with cross shape to get homozygous hosts.

After transforming stem cells non-human embryonic with the modification vector directed to alter the alpha-1,3-GT gene, the cells can be seeded on a feeding layer in a appropriate medium, for example, bovine serum enhanced DMEM fetal. The cells that contain the construct can be detected using a selective medium, and after sufficient time to for colonies to grow, colonies can be collected and analyzed for the existence of homologous recombination. May the polymerase chain reaction is used, with primers inside of and without the construction sequence but in the target locus. Those colonies that show homologous recombination after can be used for embryo manipulation and injection of blastocysts Blastocysts can be obtained from females superovulated. Embryonic stem cells can then be treated with trypsin and the modified cells added to a drop It contains the blastocysts. You can inject at least one of the modified embryonic stem cells in the blastocele of the blastocyst After the injection, at least one of the blastocysts can be returned to each of the uterine horns of Pseudopregnated females. The females are allowed to continue to term and the resulting litters are screened for mutant cells that Have the construction. The blastocysts are selected by different ancestry from transformed ES cells. Providing a different phenotype of the blastocyst and the cells ES, the chimeric progeny can be easily detected, and then genotyping can be performed to probe the presence of the gene from the modified alpha-1,3-GT.

Nuclear transfer of somatic cells to produce cloned transgenic offspring

The present invention provides a method for clone a pig that lacks a gene from the functional alpha-1,3-GT through somatic cell nuclear transfer comprising the stages listed in claim 24. In general, the pig can be produced by a nuclear transfer process that includes the following stages: obtain desired differentiated pig cells to be used as a source of donor nuclei; get oocytes from a pork; enucleate said oocytes; transfer the desired core of the differentiated cell or the cell in the enucleated oocyte, by for example, by fusion or injection, to form TN units; Activate the resulting unit of TN; and transfer said TN unit grown in a host pig so that the TN unit is Develop in a fetus.

Nuclear transfer techniques or nuclear transplant techniques are known in the art (Dai et al . Nature Biotechnology 20: 251-255; Polejaeva et al . Nature 407: 86-90 (2000); Campbell et al ., Theriogenology, 43: 181 (1995); Collas et al ., Mol. Report Dev., 38: 264-267 (1994); Keefer et al ., Biol. Reprod., 50: 935-939 (1994); Sims et al . , Proc. Natl. Acad. Sci., USA, 90: 6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, U.S. Patent Nos. 4,944,384 and 5,057 .420).

A non-human donor cell nucleus, which has been modified to alter the alpha-1,3-GT gene, is transferred to a porcine recipient oocyte. The use of this method is not restricted to a particular donor cell type. The donor cell can be as described herein, see also, for example, Wilmut et al . Nature 385 810 (1997); Campbell et al . Nature 380 64-66 (1996); Dai et al ., Nature Biotechnology 20: 251-255, 2002 or Cibelli et al . Science 280 1256-1258 (1998). All normal karyotype cells can be used, including embryonic, fetal and adult somatic cells that can be used successfully in nuclear transfer. Fetal fibroblasts are a particularly useful class of donor cells. Generally suitable methods of nuclear transfer are described in Campbell et al . Theriogenology 43 181 (1995), Dai et al . Nature Biotechnology 20: 251-255, Polejaeva et al . Nature 407: 86-90 (2000), Collas et al . Mol. Play Dev. 38 264-267 (1994), Keefer et al . Biol. Play. 50 935-939 (1994), Sims et al . Proc. Nat'l Acad. Sci. USA 90 6143-6147 (1993), WO-A-9426884; WO-A-9424274, WO-A-9807841, WO-A-9003432, U.S. Patent No. 4,994,384 and U.S. Patent No. 5,057,420. Differentiated or at least partially differentiated donor cells can also be used. The cells may also be, although not necessarily, in culture and may be quiescent. Nuclear donor cells that are quiescent are cells that can be induced to enter quiescence or that exist in a quiescent state in vivo . Prior art methods have also used embryonic cell types in cloning procedures (Campbell et al . (Nature, 380: 64-68, 1996) and Stice et al . (Biol. Reprod., 20 54: 100-110, 1996 ).

Somatic nuclear donor cells can be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disintegrated preparation of a embryo, fetus or complete adult animal or part thereof. The nuclear donor cells, for example, are selected from the group consisting of epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, heart muscle cells, other cells muscle cells, granulose cells, cumulus cells, cells epidermal or endothelial cells.

The nuclear donor cell can be a cell non-human embryonic mother. Preferably, cells can be used fibroblasts as donor cells.

The nuclear donor cells of the invention they can be germ cells of a non-human animal. Can be used any germ cell of an animal species in the phase embryonic, fetal, or adult as a nuclear donor cell. In a proper embodiment, the nuclear donor cell is a cell embryonic germ.

Nuclear donor cells can stop at any stage of the cell cycle (G0, G1, G2, S, M) to ensure coordination with the acceptor cell. Can be used any method known in the art to manipulate the phase of cellular cycle. The methods to control the cell cycle phase include, but not limited to, quiescence G0 induced by contact inhibition of cultured cells, quiescence G0 induced by the elimination of serum or other essential nutrient, G0 quiescence induced by senescence, G0 quiescence induced by the addition of a specific growth factor; quiescence G0 or G1 induced by physical or chemical means such as shock by heat, hyperbaric pressure or other treatment with an agent chemical, hormone, growth factor or other substance; control of the S phase by treatment with a chemical agent that interferes with any point of the replication procedure; M phase control by selection using separation of fluorescence activated cells, mitotic agitation, treatment with microtubule alteration agents or any agent chemical that alters progress in mitosis (see also Freshney, R. I., "Culture of Animal Cells: A Manual of Basic Technique ", Alan R. Liss, Inc, New York (1983).

Methods for oocyte isolation are well known in the art. Essentially, this may include isolating oocytes from the ovaries or the reproductive tract of a pig. An easily available source of pig eggs is slaughterhouse materials. For the combination of techniques such as genetic engineering, nuclear transfer and cloning, the oocytes generally must be matured in vitro before these cells can be used as receptor cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop in a embryo. This process generally requires collecting immature oocytes (prophase I) from mammalian ovaries, for example, bovine ovaries obtained in a slaughterhouse, and maturing the oocytes in a maturation medium before fertilization or enucleation until the oocyte reaches the metaphase phase II, which in the case of bovine oocytes usually occurs approximately 18-24 hours after aspiration. This period of time is known as the "maturation period." In certain embodiments, the oocyte is obtained from a suckling pig. A "sucker" is a female pig that has never had offspring. In other embodiments, the oocyte is obtained from a sow. A "sow" is a female pig that has previously produced offspring.

A non-human oocyte in the metaphase II phase may be the receiving oocyte, at this stage it is believed that the oocyte may be or is sufficiently "activated" to treat the introduced nucleus as a fertilizing sperm. Metaphase II phase oocytes, which have matured in vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes from non-superovulated or superovulated pigs of 35 to 48, or 39-41, hours after the onset of heat or after injection of human chorionic gonadotropin (hCG) or a similar hormone, can be surgically collected.

After a period of maturation time fixed, which varies from about 10 to 40 hours, and preferably approximately 16-18 hours, the oocytes can enucleate. Before enucleation, the oocytes can be removed and be placed in appropriate medium, such as HECM containing 1 milligram per milliliter of hyaluronidase before removing cumulus cells The oocytes separated later can scan for polar bodies, and oocytes in metaphase II selected, determined by the presence of polar bodies, is use later for nuclear transfer. Enucleation comes followed.

Enucleation can be done by methods known, such as those described in US Pat. No. 4,994,384. For example, oocytes in metaphase II can be placed in HECM, which optionally contains 7.5 micrograms per milliliter of cytochalasin B, for immediate enucleation, or can be placed in a suitable medium, for example an embryo culture medium such as CR1aa, plus 10% heat cow serum, and then enucleate later, preferably no more than 24 hours later, and more preferably 16-18 hours after.

Enucleation can be performed by microsurgery using a micropipette to remove the polar body and cytoplasm adjacent. The oocytes can then be scanned to identify those that have successfully coded. A mode of to explore the oocytes is to dye the oocytes with 1 microgram per milliliter of Hoechst 33342 dye in HECM, and then visualize the oocytes under ultraviolet irradiation for less than 10 seconds. Oocytes that have successfully enucleated then they can be sown in a suitable culture medium, by example, CR1aa plus 10% serum.

A single non-human mammalian cell of the same species can then be transferred as the enucleated oocyte into the perivithelial space of the enucleated oocyte used to produce the TN unit. The mammalian cell and the enucleated oocyte can be used to produce TN units according to methods known in the art. For example, cells can be fused by electrofusion. Electrofusion is achieved by providing a pulse of electricity that is sufficient to cause a transient decomposition of the plasma membrane. This decomposition of the plasma membrane is very short because the membrane quickly forms again. Therefore, if two adjacent membranes are induced to decompose and after the reformation the lipid bilayers intermingle, small channels can open between the two cells. Due to the thermodynamic instability of said small opening, it enlarges until the two cells become one. See, for example, U.S. Patent No. 4,997,384 to Prather et al . A variety of electrofusion media can be used including, for example, sucrose, mannitol, sorbitol and phosphate buffered solution. Fusion can also be achieved using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can be injected directly into the oocyte instead of using electroporation fusion. See, for example, Collas y Barnes, Mol. Play Dev., 38: 264-267 (1994). After fusion, the resulting fused TN units are subsequently placed in a suitable medium until activation, for example, CR1aa medium. Typically activation can be performed shortly after, for example, less than 24 hours later, or about 4-9 hours later, or optimally 1-2 hours after fusion. In a preferred embodiment, activation occurs at least one hour after fusion and 40-41 hours after maturation.

The TN unit can be activated by known methods. Such methods include, for example, cultivating the TN unit at sub-physiological temperature, in essence, applying a cold, or really icy, thermal shock to the TN unit. This can be done more conveniently by cultivating the TN unit at room temperature, which is relatively cold with respect to the physiological temperature conditions to which the embryos are usually exposed. Alternatively, activation can be achieved by the application of known activation agents. For example, the penetration of oocytes by sperm during fertilization has been shown to activate pre-fusion oocytes producing greater amounts of viable pregnancies and multiple genetically identical calves after nuclear transfer. In addition, treatments such as electric and chemical shock can be used to activate TN embryos after fusion. See, for example, U.S. Patent No. 5,496,720, to Susko-Parrish et al . In addition, activation can be performed by simultaneously or sequentially increasing the levels of divalent cations in the oocyte, and reducing the phosphorylation of cellular proteins in the oocyte. This can generally be done by introducing divalent cations into the oocyte cytoplasm, for example, magnesium, strontium, barium or calcium, for example, in the form of an ionophore. Other methods to increase divalent cation levels include the use of electric shock, ethanol treatment and treatment with caged chelators. Phosphorylation can be reduced by known methods, for example, by the addition of kinase inhibitors, for example, serine-threonine kinase inhibitors, such as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine, and sphingosine. Alternatively, phosphorylation of cellular proteins can be inhibited by the introduction of a phosphatase into the oocyte, for example, phosphatase 2A and phosphatase 2B.

The activated TN units, or "fused embryos," can then be grown in a suitable in vitro culture medium until the generation of cell colonies. Suitable culture media for embryo culture and maturation are well known in the art. Examples of known media, which can be used for embryo culture and maintenance, include Ham F-10 medium + 10% fetal calf serum (FCS), Tissue Culture Media-199 (TCM-199) + serum 10% fetal calf, Tyrode-Albumin-Lactate-Pyruvate (TALP) medium, Dulbecco phosphate buffered saline (PBS), Eagle and Whitten media and, in a specific example, activated TN units can be grown in medium NCSU-23 for about 1-4 h at about 38.6 ° C in a humidified atmosphere of 5% CO2.

Next, the cultivated unit or units of TN they can be washed and then placed in a suitable content medium in well plates preferably containing a layer of adequate confluent feeding. Feeding layers Suitable include, by way of example, fibroblasts and cells epithelial TN units are grown in the layer of feed until the TN units reach a size suitable for transfer to a receiving female, or to obtain cells that can be used to produce cell colonies. Preferably, these TN units can be grown up to minus about 2 to 400 cells, about 4 to 128 cells, or at least about 50 cells.

TN units activated later can transfer (embryo transfers) to pig oviduct female. In one embodiment, the female pigs can be suckers recipients of synchronized heat. Lineage piglets can be used crossed (large white / Duroc / Landrace) (127.01-181.44 kg (280-400 lbs)). The piglets can synchronize as receiving animals by oral administration of 18-20 mg of Regu-Mate (Altrenogest, Hoechst, Warren, NJ) mixed in the feed. Regu-Mate can be supplied for 14 days consecutive. Then they can be administered i.m. thousand units of Human Chorionic Gonadotropin (hCG, Intervet America, Millsboro, SD) approximately 105 h after the last treatment with Regu-Mate. Embryo transfers can then perform approximately 22-26 h later of hCG injection. In one embodiment, pregnancy can be carried out and produce the birth of living offspring. In another embodiment, pregnancy can stop early and may embryonic cells collected.

Breeding of desired homozygous knockout animals

A method to produce viable pigs that lack any expression of functional alpha-1,3-GT can understand cross a heterozygous male pig for the gene of the alpha-1,3-GT with a female pig heterozygous for the gene of the alpha-1,3-GT. Pigs can be heterozygous due to the genetic modification of an allele of the alpha-1,3-GT gene to avoid expression of that allele. As an alternative, pigs can be heterozygous due to the presence of a point mutation in a allele of the alpha-1,3-GT gene. The point mutation can be a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene. Be describes a method to produce a pig animal that lacks any expression of alpha-1,3-GT functional in which a male pig that contains a mutation punctual T-a-G at the second base of the exon 9 of the alpha-1,3-GT gene is crosses with a female pig that contains a point mutation T-a-G at the second base of exon 9 of the alpha-1,3-GT gene.

In one method, animals can be crossed sexually mature produced from nuclear transfer of donor cells that carry double elimination in the gene of the alpha-1,3-GT, and test your offspring for homozygous knockout. These knockout animals homozygous can then be crossed to produce more animals.

In another method, the oocytes of a sexually mature double knockout non-human animal can be fertilized in vitro using wild-type sperm from two lines of genetically different pigs and the embryos implanted in suitable substitutes. The offspring of these pairings can be tested for the presence of the elimination, for example, they can be tested by cDNA sequencing, PCR, sensitivity to toxin A and / or lectin binding. Then, in sexual maturity, the animals of each of these litters can mate.

In certain methods the pregnancies can be stopped early so that fetal fibroblasts can be isolated and further characterized phenotypically and / or genotypically. Fibroblasts that lack the expression of the alpha-1,3-GT gene can then be used for nuclear transfer according to the methods described herein (see also Dai et al .) To produce multiple pregnancies and offspring carrying the double elimination desired.

IV. Types of Modified Swine Animals Genetically

Another embodiment of the present invention provides a pig animal, in which both alleles of the gene of the alpha-1,3-GT are inactivated, by which an allele is inactivated by a modification event directed genetics and the other allele is inactivated by a mutation punctual of natural origin.

V. Organs, tissues, cells and cell lines pigs

The present invention provides, for the first time instead, viable pigs in which both alleles of the alpha 1,3 gene Galactosyltransferase have been inactivated. The invention also provides organs, tissues, and cells obtained from said pig, which are useful for xenotransplantation.

In one embodiment, the invention provides organs, tissues and / or purified cells or cell lines or substantially pure pigs obtained from pigs that lack any expression of functional alpha1,3GT.

In one embodiment, the invention provides organs that are useful for xenotransplantation. Any can be used porcine organ, including, but not limited to: brain, heart, lungs, glands, brain, eye, stomach, spleen, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, nose, mouth, lips, gums, teeth, tongue, glands salivary, tonsils, pharynx, esophagus, large intestine, intestine thin, straight, anus, pylorus, thyroid gland, thymic gland, adrenal capsule, bones, cartilage, tendons, ligaments, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testicles, seminal vesicles, penis, lymph, lymph nodes and lymph vessels.

In another embodiment, the invention provides tissues that are useful for xenotransplantation. Any can be used pig tissue, including, but not limited to: epithelium, tissue connective, blood, bone, cartilage, muscle, nerve, adenoid, adipose, areolar, bone, adipose brown, spongy, muscle, cartilaginous, cavernous, chondroid, chromaffinic, dartoic, elastic, epithelial, fatty, fibrohyaline, fibrous, Gamgee, gelatinous, granulation, lymphoid associated with the intestine, Vascular vascular, haemopoietic hard, indifferent, interstitial, of lining, islet, lymphatic, lymphoid, mesenchymal, mesonephritic, mucous connective, multilocular adipose, myeloid, soft, nephrogenic, nodal, bone, osteogenic, osteoid nasion, periapical, reticular, retiform, elastic, skeletal muscle, smooth muscle, and subcutaneous tissue.

In a further embodiment, the invention provides cells and cell lines of porcine animals that lack the expression of functional alpha1.3GT. In one embodiment, these cells or cell lines can be used for xenotransplantation. Cells of any porcine tissue or organ may be used, including, but not limited to: epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle, other muscle cells, granulose cells, cumulus cells, epidermal cells, endothelial cells, islet cells of Langerhans, pancreatic insulin secreting cells, pancreatic alpha-2 cells, beta pancreatic cells, alpha-1 pancreatic cells , blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular vascular endothelial cells, fibroblasts, hepatic star cells, muscu the aortic smooth, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, cells Thyroid, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, cells retinal, retinal canes, retinal cones, heart cells, pacemaker cells, splenic cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterus cells, prostate, vaginal epithelial cells, sperm cells, testicular cells it is, germ cells, ovules, leydig cells, peritubular cells, Sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicular cells, mucous cells, hair cells, non-keratinized epithelial cells, keratinized epithelial cells, cells pulmonary, calciform cells, columnar epithelial cells, dopaminergic cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, dopaminergics, embryonic (non-human) stem cells, fibroblasts and fetal fibroblasts. In a specific embodiment, pancreatic cells are provided, including, but not limited to, islets of Langerhans islets, insulin secreting cells, alpha-2 cells, beta cells, alpha-1 pig cells that lack the expression of alpha- 1,3-GT
functional.

Non-viable derivatives include tissues separated from viable cells by enzymatic or chemical treatment, These tissue derivatives can be further processed by crosslinking or other chemical treatments before use in transplant. In a preferred embodiment, derivatives include extracellular matrix obtained from a variety of tissues, including skin, urinary tract, bladder or submucosal tissues of the organs. In addition, tendons, joints and separate bones of viable tissue to include the valves Cardiac and other non-viable tissues such as medical devices.

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Therapeutic uses

Cells can be administered in a host upon request in a wide variety of ways. Preferred modes of administration are parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrasternal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intramuscular, intranasal, subcutaneous, intraorbital, intracapsular, topical, per patch. transdermal, by rectal, vaginal or urethral administration including by suppository, percutaneous, by nasal spray, by surgical implant, internal surgical patch, infusion pump, or by catheter. In one example, the agent and the vehicle are administered in a slow release formulation such as a direct injection into the tissue or in embolate, implant, microparticle, microsphere, nanoparticle or
nanosphere

Disorders that can be treated by infusion of the described cells include, but are not limited to, diseases resulting from a failure or a dysfunction of normal blood cell production and maturation (ie aplastic anemia and hyperproliferative stem cell disorders); neoplastic, malignant diseases in the hematopoietic organs (for example, leukemia and lymphomas); malignant solid tumors of broad spectrum of nonhematopoietic origin; autoimmune conditions; and genetic disorders. Such disorders include, but are not limited to, diseases resulting from failure or dysfunction of normal blood cell production and maturation, hyperproliferative stem cell disorders, including aplastic anemia, pancytopenia, agranulocytosis, thrombocytopenia, red blood cell aplasia, Blackfan syndrome. Diamond, due to drugs, radiation, or idiopathic infection; hematopoietic malignancies including acute lymphoblastic (lymphocytic) leukemine, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute malignant myelosclerosis, multiple myeloma, vera polycythemia, agnogenic myelometaplasia, Waldenstrom macroglobulinemia, Hodgodkin lymphoma; immunosuppression in patients with malignant solid tumors including malignant melanoma, stomach carcinoma, ovarian carcinoma, breast carcinoma, small cell lung carcinoma, retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma, lymphoma; autoimmune diseases including rheumatoid arthritis, type I diabetes, chronic hepatitis, multiple sclerosis, systemic lupus erythematosus; genetic disorders (congenital) including anemias, familial aplastic, Fanconi syndrome, deficiencies of dihydrofolate reductase, deficiency of formamino transferase, Lesch-Nyhan syndrome, congenital dyseritropoietic syndrome I-IV, Chwachmann-Diamond syndrome, dihydrofolate deficiencies reductase, formamino transferase deficiency, Lesch-Nyhan syndrome, congenital spherocytosis, congenital elliptocytosis, congenital stomatocytosis, congenital Rh null disease, paroxysmal nocturnal hemoglobinuria, variants 1, 2, 3 of the G6PD (glucose-6-phosphate dehydrogenase) , pyruvate kinase deficiency, congenital sensitivity to erythropoietin, deficiency, disease and sickle cell traits, thalassemia alpha, beta, gamma, methemoglobinemia, congenital immunity disorders, severe combined immunodeficiency disease (SCID), naked lymphocyte syndrome, sensitive combined immunodeficiency to ionophores, immunodeficiency combined with u na coating abnormality, nucleoside phosphorylase deficiency, granulocyte actin deficiency, childhood agranulocytosis, Gaucher disease, adenosine deaminase deficiency, Kostmann syndrome, reticular dysgenesis, congenital leukocyte dysfunction syndromes; and others such as osteoporosis, myelosclerosis, acquired hemolytic anemias, acquired immunodeficiencies, infectious disorders that cause primary or secondary immunodeficiencies, bacterial infections (e.g., Brucellosis, Listeriosis, tuberculosis, leprosy), parasitic infections (e.g., malaria, Leishmaniasis), fungal infections, disorders involving disproportion in series of lymphoid cells and immune functions altered due to age, phagocytic disorders, Kostmann agranulocytosis, chronic granulomatous disease, Chediak-Higachi syndrome, neutrophil actin deficiency, GP-180 deficiency of the neutrophil membrane, metabolic storage diseases, mucopolysaccharidosis, mucolipidosis, various disorders involving immune mechanisms, Wiskott-Aldrich syndrome, alpha 1-antitrypsin deficiency,
etc.

Diseases or pathologies include neurodegenerative diseases, hepatodegenerative diseases, nephrodegenerative diseases, spinal cord injury, trauma or cranial surgery, viral infections that cause degeneration of tissues, organs, or glands, and the like. Such neurodegenerative diseases include, but are not limited to, AIDS dementia complex; demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders, such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders, such as Huntington's chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs that block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; progressive supra-nuclear paralysis; structural lesions of the cerebellum; spinocerebelar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple system degenerations (Mencel, Dejerine Thomas, Shi-Drager, and Machado-Joseph), systemic disorders, such as Rufsum 'disease, abetalipoproteinemia, ataxia, telangiectasia; and multisystem mitochondrial disorder; central demyelinating disorders, such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit, such as neurogenic muscular atrophies (cellular degeneration of the anterior horn, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer disease; Down syndrome at middle age; disease with diffuse Lewy bodies; senile dementia of type Lewy bodies; Parkinson's disease, Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis, Hallervorden-Spatz disease; and pugilistic dementia. See, for example, Berkow et al ., (Eds.) (1987), The Merck Manual, (15a) ed.), Merck and Co., Rahway,
NJ.

The present invention is described in detail. Additional in the following examples. It is intended that the examples provided below are illustrative only, and are not It is intended to limit the scope of the invention.

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Examples

Example one

Production of Heterozygous Swine Cells for the gene of the alpha-1,3-GT

Isolation and transfection of primary porcine fetal fibroblasts . Fetal fibroblast cells (PCFF4-1 to PCFF4-10) were isolated from 10 fetuses of the same pregnancy on day 33 of gestation. After removing the head and viscera, the fetuses were washed with Hank's balanced salt solution (HBSS; Gibco-BRL, Rockville, MD), placed in 20 ml of HBSS, and sliced with small surgical scissors. The tissue was pelleted and resuspended in 50 ml tubes with 40 ml of DMEM and 100 U / ml collagenase (Gibco-BRL) per fetus. The tubes were incubated for 40 min. in a shaking water bath at 37 ° C. The digested tissue was allowed to stand for 3-4 min. and the cell-rich supernatant was transferred to a new 50 ml tube and sedimented. The cells were then resuspended in 40 ml of DMEM containing 10% fetal calf serum (FCS), 1X non-essential amino acids, 1 mM sodium pyruvate and 2 ng / ml bFGF, and seeded in 10 cm plates. All cells were cryopreserved after reaching confluence. SLA-1 to SLA-10 cells were isolated from 10 fetuses on day 28 of pregnancy. The fetuses were crushed through a 60 mesh metal sieve using slowly curved surgical forceps so as not to generate excessive heat. The cell suspension was then pelleted and resuspended in 30 ml of DMEM containing 10% FCS, 1X nonessential amino acids, 2 ng / ml bFGF, and 10 µg / ml gentamicin. The cells were seeded on 10 cm plates, grown for one to three days, and cryopreserved. For transfections, 10 µg of linearized vector DNA was introduced into 2 million cells by electroporation. Forty-eight hours after transfection, the transfected cells were seeded in 48-well plates at a density of 2,000 cells per well and selected with 250 µg / ml of G418.

Construction of the knockout vector . Two knockout vectors for alpha-1,3-GT, pPL654 and pPL657, were constructed from isogenic DNA of two primary porcine fetal fibroblasts, SLA1-10 and PCFF4-2 cells. A 6.8 kb genomic fragment of alpha-1,3-GT, which includes most of intron 8 and exon 9, was generated by PCR from purified DNA from SLA1-10 cells and PCFF4-2 cells, respectively. The unique EcoRV site at the 5 'end of exon 9 was converted into a SalI site and a 1.8 kb fragment IRES-neo-poly A was inserted into the SalI site. IRES (internal ribosome entry site) functions as the translation initiation site for the neo protein. Thus, both vectors have a 4.9 kb 5 'recombination arm and a 1.9 kb 3' recombination arm
(Figure 6).

3 'PCR and long range PCR . Approximately 1,000 cells were resuspended in 5 µl of embryo lysis buffer (ELB) (40 mM Tris, pH 8.9, 0.9% Triton X-100, 0.9% NP40, 0.4 mg / ml of proteinase K), were incubated at 65 ° C for 15 min. to lyse the cells and were heated to 95 ° C for 10 min. to inactivate proteinase K. For 3 'PCR analysis, the fragments were amplified using the high fidelity expansion PCR system (Roche Molecular Biochemicals) in 25 µl of reaction volume with the following parameters: 35 cycles of 1 min. at 94 ° C, 1 min. at 60 ° C, and 2 min. at 72 ° C. For the LR-PCR, the fragments were amplified using the TAKARA LA system (Panvera / Takara) in 50 µl of reaction volume with the following parameters: 30 cycles of 10 s at 94 ° C, 30 s at 65 ° C, 10 min. + 20 s increase / cycle at 68 ° C, followed by a final cycle of 7 min. at 68 ° C. The 3 'PCR and LR-PCR conditions for the purified DNA were the same as for the cells except that 1 µl of purified DNA (30 µg / ml) was mixed with 4 µL of ELB.

Southern blot analysis of cell samples . Approximately 10 6 cells were lysed overnight at 60 ° C in lysis buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% Sarcosil (w / v), 1 mg / ml of proteinase K) and the DNA was precipitated with ethanol. The DNA was then digested with BstEII and separated on a 1% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane and probed with the probe labeled with digoxigenin at the 3 'end. Bands were detected using a chemiluminescent substrate system (Roche Molecular Biochemicals).

Results Antibiotic resistant colonies (G418) were screened by 3 'PCR with neo442S and αGTE9A2 as direct and reverse primers. Neo442S is at the 3 'end of the neo gene and αGTE9A2 is at the 3' end of exon 9 in sequences located outside the 3 'recombination arm (Figure 6). Therefore, only the expected 2.4 kb PCR product would be obtained through satisfactory targeted modification in the? 1,3,3TT locus. From a total of seven transfections in four different cell lines, 1105 G418 resistant colonies were collected, of which 100 (9%) were positive for the alteration of the α1.3 GT gene in the initial 3 'PCR scan (interval 2.5-12%). Colonies 657A-A8, 657A-I6, and 657A-I11 showed the expected 2.4 kb band, while control PCFF4-6 cells, and another colony resistant to G418, 657A-P6, were negative. A portion of each colony positive to 3 'PCR was frozen immediately, in several small aliquots, for future use in TN experiments, while the rest of the cells were expanded for long-range PCR (LR-PCR) and transfer of Southern

As the PCR analysis to detect junctions of recombination, or mRNA analysis (RT-PCR) can generate false positive results, a long PCR was performed scope, which would cover the modified region in a targeted manner complete. The LR-PCR covers the genomic sequence of 7.4 kb of α1.3GT from exon 8 to the end of exon 9, with the two primers (αGTE8S and αGTE9A2) located outside the recombination region (Figure 2). The PCFF4-6 control cells, and the negative colony to 3 'PCR, 657A-P6, showed only the band of 7.4 kb endogenous of the wild type α1.3GT locus. In contrast, three of the 3 'PCR positive colonies, 657A-A8, 657A-I6 and 657A-I11, showed both the endogenous band 7.4 kb as a new 9.2 kb band, of the expected size for the Directed insertion of the 1.8 kb IRES-neo cassette in the α1,3GT locus.

Approximately half (17/30) of the colonies LR-PCR positively expanded successfully to produce sufficient numbers of cells (1 x 10 6 cells) for Southern analysis. It was anticipated that the colonies would be heterozygous for the knockout in the α1,3 GT locus, and by both must have a copy of the unmodified normal gene, and a altered copy of the α1,3 GT gene. With digestion with BstEII, the Knockout cells for α1,3 GT must show two bands: one 7 kb band of expected size for the α1.3 GT allele endogenous, and a 9 kb band characteristic of the insertion of IRES-neo sequences in the α1,3 GT locus (Figure 2). The 17 colonies positive for LR-PCR are confirmed by Southern analysis for the knockout. The same membranes were re-probed with specific sequences for neo and the 9 kb band was detected with the neo probe, forming this directed insertion mode of the IRES-neo cassette in the  altered α1,3GT locus.

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Example 2

Production of homozygous swine cells for the gene of the alpha-1,3-GT

Fetal knockout fibroblasts for heterozygous alpha-1,3-GT cells (657A-I11 1-6) were isolated from a pregnancy on day 32 as described above (See also Dai et al . Nature Biotechnology 20: 451 ( 2002)). A knockout vector for alpha-1,3-GT directed to ATG (start codon) (pPL680), which also contained a neo gene, was constructed to eliminate the second allele of the alpha-1,3-GT gene. These cells were transfected by electroporation with pPL680 and were selected for the alpha-1,3Gal-negative phenotype with purified C. difficile toxin A (described below).

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Example 3

Toxin A selection of C. difficile from Homozygous Swine Cells for the alpha-1,3-GT gene Toxin A cytotoxicity curve

Swine cells (PCFF4-6) were exposed for 1 hour or overnight at dilutions in series of factor ten toxin A (0.00001 µg / ml at 10 \ mug / ml). The cells were grown in 24-well plates and were incubated with the toxin for 1 hour or overnight at 37 ° C. The results of this exhibition are detailed in Table 2. Clearly, a 1-hour exposure to toxin A at> 1 µg / ml caused a cytotoxic effect in> 90% of the cells. For the both a concentration of toxin A was chosen a or slightly by above 1 µg / ml for the selection of altered cells genetically

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TABLE 2 Toxicity of toxin A at 1 hour exposure and overnight

6

Disintegrated cells of a porcine embryo (I-11: 1-6) containing a previously identified directed knockout in an allele of the alpha-1,3-GT gal gene (Dai et al .) Were transfected with 10 µg of linearized vector DNA (promoter capture) by electroporation. After 48 hours, the cells were seeded in 48-well plates at a density of 2000 cells per well and selected with 250 µg / ml of G418. Five days after transfection, the medium was removed from the wells, and replaced with 2 µg / ml toxin A in culture medium (high glucose DMEM with 2.8 µg / ml bFGF and FCS to 20%). The cells were exposed to the selective effect of toxin A for 2 hours at 37 ° C. The medium containing toxin A was removed, along with any affected cells that had been released from the plaque surface, the remaining cells were washed with fresh medium, and the medium was replaced without toxin A. Ten days later, the cells were again exposed to toxin A at 1.3 µg / ml in medium for 2 hours at 37 ° C. The medium, toxin A, and any solution cells were removed, the remaining cells were washed, and the medium was replaced.

Sixteen days after transfection, it collected a single colony that showed insensitivity to toxin A, called 680B1, and a part was sent for DNA analysis and lectin staining. DNA analysis indicated that the insensitivity to toxin A was not due to the integration of the second target vector; however, the cells were not stained with GSL lectin IB-4, indicating that a knockout had happened Functional locus. 680B1 double knockout cells were used for nuclear transfer in 5 receptors and three were produced you pregnan Two of these pregnancies suffered an abortion spontaneously in the first month; the four fetuses of the remaining pregnancy are collected on day 39 of pregnancy and the cells disintegrated and planted in tissue culture. These fetal cells (680B1-1, 680B1-2, 680B1-3, 680B1-4) were exposed to toxin A at 1 mug / ml for 1 hour at 37 ° C, followed by media removal, cell wash, and media replacement without toxin A. Fetuses 1, 2, and 4 were not affected by the toxin A, while most of the cells of the fetus 3 are rounded, indicating that this embryo was sensitive to cytotoxic effects of toxin A.

Fetuses 1, 2, and 4 did not bind to GS lectin IB4, as indicated by the FACS analysis (see Table 3), while 3 joined lectin. This suggests that fetuses 1, 2, and 4 do not carry the 1.3 gal alpha epitope for which this lectin particular is specific.

TABLE 3 Results of 680B1-1 cell FACS to 680B1-4 with lectin GS-IB4

GS IB4 lectin positive cells (%)

7

A complement fixation test was performed on cells of the four fetuses. The lysis test of complement was developed as a bioassay for the absence of alpha gal expression. Human serum contains high levels of preformed antibodies against alpha gal as well as the repertoire full of complement regulatory proteins (the C3 pathway). The presence of alpha gal on the surface of a cell, after the binding of the anti-alpha gal antibody activates the complement cascade, and causes cell lysis mediated by the complement. The negative alpha-gal cells would be resistant to complement-mediated lysis. In three trials different, B1 pig and control cells were exposed to serum human plus complement, and trials were conducted to evaluate the sensitivity or resistance to cell lysis mediated by complement indicated by alpha-gal. The essay is performed with cells B1-1, B1-2, and B1-4, as well as heterozygous KO GT cells (B1-3, positive gal), and with pig cells PCFF4-6 alpha-gal (+) type wild as control. The cells were exposed to one of three treatments; two negative controls, bovine serum albumin (BSA), and heat inactivated human serum (HIA-HS) no they contain no functional complement protein and therefore not would be expected to cause no significant cell lysis; he third treatment, non-heat inactivated human serum (NHS) contains the functional human complement as well as antibodies specific anti-gal, and therefore would be expected to lisara cells that have galactose alpha 1,3 galactose on its cell surface

The results shown in Figure 1 clearly demonstrate that B1-1 cells, B-2 and B1-4 are resistant to lysis mediated by human complement while cells B1-3, which are α1.3 Gal positive, are still as sensitive to human plasma as cells PCFF4-6 wild type.

The cDNA sequencing results of all fetuses indicated that fetuses 1, 2 and 4 contain a point mutation in the second alpha 1.3 GT allele, a change that It could produce a dysfunctional enzyme (see Figure 2). This mutation was happening at bp 424 of the coding region, significantly, the second base pair of exon 9, of the gene of Alpha-1,3-GT (GGTA1) (Accession No. to GenBank L36152) as a conversion of a thymine residue to guanine, which causes an amino acid substitution of tyrosine in the aa 142 in an aspartic acid.

This is a significant conversion, since tyrosine, a hydrophilic amino acid, is a critical component of the UDP binding site of alpha 1.3GT (see Fig. 3). Analysis of the crystalline structure of bovine alpha-1,3-GT protein showed that this tyrosine is the center of the enzyme's catalytic domain, and is involved in UDP-Gal binding (Gastinel et al ., EMBO Journal 20 ( 4): 638-649, 2001). Therefore, a change of tyrosine (a hydrophobic amino acid) into aspartic acid (a hydrophilic amino acid) would be expected to cause the alteration of the αGT function (as noted).

To confirm that the mutated cDNA will not create functional αGT protein, the cDNAs of the second alleles of the 4 cells were cloned into an expression vector and this GT expression vector was introduced by transfection into human fibroblast cells (HeLa cells) as well as in primary Rhesus monkey cells. Since humans and old-world monkeys lack the functional alpha 1.3 GT gene, HeLa cells would not have an alpha 1.3 galactose on their cell surface (as tested by lectin binding experiments). The results showed that HeLa and monkey cells, when transfected with cDNA obtained from B1-1, B1-2 and B1-4 cells, were still negative α1.3 Gal by staining with IB4-lectin, while Hela and of Rhesus monkey transfected with B1-3 cell cDNA, created a functional transcript of alpha 1.3 GT and subsequently were α1.3Gal positive. Clearly, cells with the aspartate mutation (instead of tyrosine) cannot create alpha 1,3 galactosyltransferase
functional.

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Example 4

Generation of Cloned Pigs Using Fetal Fibroblasts Deficient in Alpha 1.3 GT homozygous as Donors Nuclear

Preparation of cells for nuclear transfer. Donor cells were genetically engineered to produce homozygous cells for alpha 1.3 GT deficiency as described in general lines above. Nuclear transfer was performed by methods that are well known in the art (see, for example, Dai et al ., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et al ., Nature 407: 86-90, 2000) , using porcine fibroblasts selected with toxin A as nuclear donors that were produced as described in detail hereinbefore.

Embryo transfer and live births resulting. In the initial attempt to produce live pigs alpha-1,3-GT dKO per transfer nuclear, a total of 16 embryo transfers were made with genetically manipulated donor cells. Nine were established initial pregnancies but only two reached beyond day 75 of gestation. Five piglets were born on July 25, 2002. A piglet died immediately after birth and another four They were born alive and looked normal (Figure 4).

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Example 5

Homozygous alpha 1.3 GT knockout pig analysis

Fibroblast cells of the tail and umbilical tissue sections of the 5 double knockout piglets were obtained and stained using GS-IB4 lectin as previously described. No staining was observed, indicating a complete absence of the galactose alpha 1,3 galactose epitope on the tissue surface of these animals (data not shown). Endothelial cells of the aorta and muscle fibroblasts and the tail of the dead piglet (761-1) were negative with GS-IB4 lectin staining. FACS analysis of piglet muscle fibroblasts 761-1 also showed a negative result for GS-IB4 binding. Tissue sections of the liver, kidney, spleen, skin, intestine, muscle, brain, heart, pancreas, lung, aorta, tongue, belly button, and tail obtained from piglet 761-1 were all negative with GS-IB4 staining, indicating a complete absence of detectable 1,3Gal alpha cell surface epitopes (Phelps et al ., Science 299: 411-414, 2003 including Figure S3).

An immunogenicity test was performed in vivo with knockout mice for alpha 1.3GT. Islet-like cell groups (ICC) isolated from the pancreas of the piglet 761-1 were injected intraperitoneally into knockout mice for alpha 1.3GT. CCI of a wild-type baby piglet was used as a control. As shown in fig. 5, no increase in immunoglobulin M (IgM) titer against alpha 1.3 gal was observed in knockout mice for alpha 1.3GT after ICC injection of the piglet alpha 1.3GT DKO, in contrast to significant increases in IgM titer observed in those mice injected with wild-type piglet ICC (Phelps et al ., Science 299: 411-414, 2003 including Figure S4). This result clearly demonstrates that DKO piglet cells do not create any 1,3Gal alpha epitope.

DNA sequencing obtained from the five piglets confirmed the presence of the mutation in pb 424 of the gene GGTA1, as seen in the 680B1-2 cells used to clone these animals (Figure 2).

From this first satisfactory production of a litter of alpha-GT dKO pigs have been produced two subsequent litters of dKO piglets by nuclear transfer, in one case (litter 662) using dKO fetal fibroblasts as nuclear donor cells Litter 660 was produced by nuclear transfer using fibroblast cells from the tail of a tail member of litter 761 as a nuclear donor. These births are summary in Table 4.

TABLE 4 Summary of double knockout births for alpha-GT produced by transfer nuclear

8

Example 6

Crossing male and female simple knockout pigs (SKO) for alpha 1.3 heterozygous GT to establish a pig minipiara double knockout (DKO)

A total of 29 female pigs have been generated GT-SKO clones confirmed by transfer of Southern and 25 cloned GT-SKO male pigs confirmed by Southern transfer to date. These heterozygous males and females (simple knockout pigs for gene of the alpha1,3GT) have been crossed by natural crossing and by artificial insemination (AI), to generate a DKO pig herd for use in preclinical studies and human clinical trials. Be have produced 16 piglets alpha1,3-GT DKO of 13 litters

This invention has been described with reference to illustrative embodiments. Other embodiments of the invention general described in this document and modifications will be evident for specialists in the art and all are considered within of the scope of the invention.

Claims (49)

1. A pig in which both alleles of the gene of the alpha-1,3-GT are inactive, where a allele becomes inactive through at least one mutation punctual. 2. A pig according to claim 1, in which at least one allele becomes inactive due to an event  of directed genetic modification. 3. A pig according to claim 2, where the directed genetic modification event is homologous recombination. 4. A pig according to claim 2 or claim 3, wherein the genetic modification event directed uses a modification construct directed to exon 9 of the alpha-1,3-GT gene. 5. A pig according to any one of the claims 1 to 4, wherein both alleles become inactive through at least one point mutation. 6. A pig according to any one of the claims 1 to 5, wherein the at least one point mutation is a substitution, deletion or insertion mutation or a combination from the same. 7. A pig according to any one of the claims 1 to 6, wherein at least one point mutation is a point mutation T-a-G in the second exon base 9. 8. A pig according to claim 7, which includes the point mutation T-a-G at the second base of exon 9 in both alleles of the gene of the alpha-1,3-GT. 9. A pig according to any one of the claims 1 to 8, wherein an allele becomes inactive through of at least two point mutations. 10. An organ of a pig according to a any of claims 1 to 9. 11. The organ of claim 10, wherein the organ is a kidney, a liver, a heart, a lung or a pancreas. 12. A tissue of a pig according to a any of claims 1 to 9. 13. The fabric of claim 12, wherein the tissue is cartilage, bone, adipose or muscle. 14. A cell or cell line of a pig according to any one of claims 1 to 9. 15. The cell of claim 14, wherein the cell is obtained from the pancreas. 16. The cell of claim 15, wherein the cell is an islet cell of Langerhans or a cell insulin secretor 17. A method to select cells that they lack the expression of galactose alpha-1,3 galactose, which comprises:

to)

exposing a population of cells to Clostridium difficile toxin A;

b)

remove the cells that rise from the surface matrix because they are adversely affected by toxin A due to receptor-mediated cytotoxicity of the toxin; Y

C)

expand and maintain those cells that do not show the cytotoxicity effects measured by receiver.

18. The method of claim 17, wherein the Clostridium difficile toxin A used for selection is in the form of a purified toxin. 19. The method of claim 17, wherein the Clostridium difficile toxin A used for selection is in the form of a culture supernatant fluid. 20. The method of claim 17, wherein the purified toxin is applied to dispersed cells, and wherein said dispersed cells are then grown in vitro in containers suitable for cell adhesion. 21. The method of claim 17, wherein The purified toxin is applied to adhered cells. 22. The method of claim 17, wherein the culture supernatant fluid is applied to dispersed cells and not adhered followed by cultivation in containers suitable for cell adhesion 23. The method of any one of the claims 17 to 22, wherein the cells are cells swine 24. A method to produce a non-human animal deficient in alpha 1.3 GT comprising:

to)

select nonhuman cells that they lack the expression of functional alpha-1,3-GT performing a method according to any one of claims 17 to 2. 3;

b)

use the toxin A resistant cells selected in step (a) as nuclear donors for nuclear transplantation in a cell neither adequate human recipient;

C)

implant the fused cells and activated in a substitute non-human female; Y

d)

produce a cloned animal that shows a deficiency or complete absence of epitopes gal alpha 1,3-gal on its cell surfaces.

25. The method of claim 24, wherein both alleles of the gene of the alpha-1,3-GT in cells a select are inactive, and an allele becomes inactive through of at least one point mutation. 26. The method of claim 25, wherein at least one allele of the gene of the alpha-1,3-GT in cells a select has become inactive due to an event of targeted genetic modification. 27. The method of claim 25 or claim 26, wherein both alleles of the alpha-1,3-GT in cells a select become inactive through at least one mutation punctual. 28. The method of any one of the claims 25 to 27, wherein the point mutation is a replacement mutation, deletion or insertion or a combination of the same. 29. The method of any one of the claims 25 to 28, wherein at least one point mutation is  a point mutation T-a-G in the second base of exon 9. 30. The method of claim 29, wherein the cells to be selected comprise the point mutation T-a-G at the second base of exon 9 in both alleles of the gene of the alpha-1,3-GT. 31. The method of claim 24, wherein the cells to be selected have become homozygous with respect to the expression of the 1,3-GT alpha allele, by directed knockout of both alleles by homologous recombination. 32. The method of claim 24, wherein the cells to be selected are homozygous with respect to the expression of the 1,3-GT alpha allele achieved by natural mutations of both alpha alleles 1,3-GT, which disables the alpha gene 1,3-GT. 33. The method of claim 24, wherein the cells to be selected are homozygous with respect to the expression of the 1,3-GT alpha allele, achieved by directed knockout of an allele by homologous recombination and natural mutation of the second allele that disables the alpha gene 1,3-GT. 34. The method of claim 24, wherein the cells to be selected have become homozygous with respect to the expression of the 1,3-GT alpha allele by induced mutations of both alpha alleles 1,3-GT, which disables the alpha gene 1,3-GT. 35. The method of claim 24, wherein This animal is a pig. 36. The method of claim 24, wherein a mutation is induced by a mutagenic agent selected from the group consisting of a chemical mutagen, radiation, and a transposon 37. The method of claim 24, wherein said chemical mutagen is selected from the group consisting of EMS, ENU, mustard gas and ICR191. 38. The method of claim 24, wherein said radiation is selected from the group consisting of ultraviolet radiation, alpha radiation, beta radiation and radiation gamma 39. A cell in which both alleles of its gene of the alpha-1,3-GT are inactive in which at least one allele contains a natural or spontaneous mutation in the alpha-1,3-GT gene, where said cell can be produced by a method comprising:

to)

expose a population of cells to C. difficile toxin A;

b)

remove the cells that have been seen adversely affected by toxin A due to cytotoxicity  toxin receptor mediated; Y

C)

expand and maintain a cell that doesn't shows the effects of receptor-mediated cytotoxicity.

40. The cell of claim 39, wherein at least one allele contains the substitution of thymine bases to guanine at the position of base 424 of the alpha gene 1,3-GT, which causes an amino acid substitution from tyrosine to aspartic acid at position 142 in the protein alpha-1,3-GT. 41. The cell of claim 39, wherein at least one allele contains an induced mutation in the gene of the alpha-1,3-GT. 42. A non-human animal that can be produced from according to the method of claim 24, wherein both alleles of the alpha-1,3-GT gene are inactive 43. A non-human animal that can be produced by cloning nuclear transfer using the cell of a any of claims 39 to 41 as a nuclear donor. 44. A cell, tissue, or organ that can be obtained from the animal of claim 42 or claim 43, or from a pig according to any one of claims 1 to 9, for use as a supplement or replacement in vivo or ex alive for cells, tissues or recipient organs. 45. A recombinant construction to modify the endogenous sequence of the gene of the alpha-1,3-GT of the pig, in which the sequence comprises a point mutation T-a-G at the second base of the exon 9. 46. A recombinant construction according to claim 45, wherein the gene sequence of the alpha-1,3-GT comprises at least 50 contiguous nucleotides 47. A method to produce cells that are heterozygous for alpha-1,3-GT, comprising transfection of porcine fetal fibroblasts primary with a construction according to any one of the claims 45 to 47. 48. A pig cell in which both alleles of the alpha-1,3-GT gene are inactive, where an allele becomes inactive through at least a timely mutation 49. A pig cell in which at least one alpha 1,3-GT allele contains a substitution of thymine base to guanine in position of base 424, which causes amino acid substitution of tyrosine to aspartic acid in the position 142 in the protein alpha-1,3-GT.